Prenatal screening

ABSTRACT

The present invention concerns products and methods Particularly useful for activating and analyzing non-dividing cell nuclei. The featured products include activating egg extracts, cytostatic factor (CSF) extracts, kits containing these extracts, and a microchamber microscope slide useful in analyzing nucleus activation.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/798,061, filedMar. 10, 2004, which is a continuation of U.S. Ser. No. 09/226,766,filed Jan. 6, 1999, now U.S. Pat. No. 6,753,457, which is a continuationof U.S. Ser. No. 09/050,380, filed Mar. 30, 1998, which is acontinuation of U.S. Ser. No. 08/455,981, filed May 31, 1995, now U.S.Pat. No. 5,773,217, which is a division of U.S. Ser. No. 08/190,771,filed Feb. 1, 1994, now U.S. Pat. No. 5,651,992, which is acontinuation-in-part of U.S. Ser. No. 08/013,039, filed Feb. 3, 1993,now U.S. Pat. No. 5,480,772, each of which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention concerns products, methods, and apparatus for analysis ofnon-dividing mammalian cell nuclei, such as human fetal cell nuclei andmammalian sperm cell nuclei.

BACKGROUND OF THE INVENTION

Jackson, Seminars in Perinatology 15: 49 (1991, describes variousprocedures to diagnose diseases. These procedures involve analysis ofthe DNA present in early embryonic states. Specifically, Jacksonmentions the use of a polymerase chain reaction to amplify genes, andthe possibility of testing oocytes by polar body assay. According toJackson:

“There are other conceivable embryo biopsy approaches for prenataldiagnosis. The trophectoderm may be obtained at later, multicellularembryonic stages when more cells might be obtained and induced toreplicate in tissue culture . . . . Another approach to early prenataldiagnosis is the recovery of fetal cells in the maternal circulation.This tantalizing possibility for a non-invasive method has been pursuedfor several years by groups in both the United States and the UnitedKingdom. Both groups originally sought placental immunologic markers foridentification and recovery of these cells. Several trophoblastantibodies were developed, some of which appeared to have relativespecificity for the fetal cell. After sporadic reports of success,recent articles appear to indicate that these markers are insufficientlyspecific and actually are attached to maternal cells frequently enoughto make this approach unworkable to date.”

Bianchi et al., Proc. Natl. Acad. Sci. USA 87: 3279 (1990), describeisolating fetal nucleated erythrocytes in maternal blood using amonoclonal antibody against the transferrin receptor. They state thatthey “were successful in detecting the Y chromosomal sequence in 75% ofmale-bearing pregnancies, demonstrating that it is possible to isolatefetal gene sequences from cells in maternal blood.”

According to Roberts, Science 18: 378 (1991), two procedures availablefor prenatal screening are chorionic villus sampling (CVS) andamniocentesis. Both these procedures have problems involving waitingtime and risk of miscarriage, “estimated at 1% to 2% for CVS and 0.5%for amniocentesis.” Supra. Roberts also points out a procedure foranalyzing nuclear DNA directly when cells are in interphase.

Lohka and Masui, Science 220: 719 (1983), describe inducing theformation of a nuclear envelope in demembraned sperm of Xenopus laevisusing a cell-free preparation from the cytoplasm of activated eggs ofRana pipiens.

Leno and Laskey, J. Cell Biology 112: 557, (1991), performed experimentsusing erythrocytes from adult chickens. According to Leno:

“Coppock et al. (1989) [Supra] have reported that a pretreatment withtrypsin was required for nuclear decondensation and DNA replication ofXenopus erythrocyte nuclei in egg extract. Trypsin pretreatment was notrequired for nuclear decondensation and DNA replication in ourextracts.”

Gordon et al., Experimental Cell Research 157: 409 (1985), describe “asystem for the activation of human sperm using cell-free extracts fromXenopus laevis eggs.” Similarly, an abstract, by Brown et al., J. CellBiology 99: 396a (1984), indicate that nuclear changes which occurduring the early phases of fertilization can be stimulated by injectingisolated sperm nuclei into heterologous recipient eggs, or by incubatingfrog sperm nuclei in the presence of cell-free extracts from frog eggs.They state that they found human sperm can be activated in vitro usingXenopus laevis frog egg extract to stimulate the early events of nuclearactivation, including chromatin decondensation, nuclear enlargement andDNA synthesis.

SUMMARY OF THE INVENTION

The present invention concerns products and methods useful for causingnon-dividing nuclei to activate (e.g., go through one or more steps ofnuclear activation). The featured products and methods are particularlyuseful for activating human fetal cell nuclei and mammalian sperm cellnuclei. “Activation” of a non-dividing cell nucleus refers to one ormore of the following activities: nuclear swelling, nucleic acidreplication, and nuclear entrance into mitosis thereby producingmetaphase chromosomes (arrested metaphase chromosomes or replicatingchromosomes). Complete activation refers to activation wherein all ofthe activities occur.

Nucleic acids can be analyzed at the different stages of activation,brought about by the present invention, to obtain useful informationsuch as information about nucleic acid structure, sequences, number ofcopies of a nucleic acid sequence, and nuclear location of a nucleicacid. Analysis of nucleic acids can be carried out using techniquesknown in the art such as in-situ hybridization and karyotype analysis ofmetaphase chromosomes.

One particular advantage of the present invention is its use in prenataldiagnosis. Activation of fetal cell nuclei can be used to facilitateprenatal diagnosis of various human conditions. Nuclei from of all typesof human fetal cells including blood cells (such as red cells, whitecells and other circulating cells of the fetus), as well as other typesof fetal cells such as cells found in the amniotic fluid, or cellsderived from the placenta (such as trophoblasts or syncytialtrophoblasts), can be activated using the described products andmethods. Preferably, the fetal cells to be activated are recovered fromthe blood or tissue of a pregnant woman rather than directly from thefetus or placenta, thereby decreasing the likelihood of discomfort orharm to the fetus and/or mother by the diagnosis procedure.

“Activation activity” refers to the ability of an agent to bring aboutnuclear activation. Examples of agents which bring about nuclearactivation include a non-activated cytostatic factor (CSF) extract andactivating egg extract. Enhancement of activation activity refers to anincrease in the activation activity which is brought about by an agentwhich causes nuclear activation. Examples of agents which enhancenuclear activation caused by an activating agent include CSF extract,purified components thereof, and proteases.

Activation activity can be measured using techniques known in the art.Such techniques include microscopic visualization of swollen nuclei,incorporation of labelled nucleic acid precursors into newly synthesizednucleic acid, microscopic visualization of metaphase chromosomes, and insitu hybridization.

The featured methods include pretreating a non-dividing human nucleus toenhance its ability to activate, bringing about complete or partialnuclear activation, and both bringing about and analyzing such nuclearactivation on a microchamber microscope slide. Other useful methodsdisclosed include preparing products such as an activating egg extract,a CSF extract, and a modified CSF extract; the use of a proteasepretreatment step in the activation of sperm; an activation assay; aretroviral integration assay; and a procedure for cloning whole animalsusing activated nuclei.

The featured products including activating egg extract, CSF extract,kits containing these extracts, and a microchamber microscope slideuseful in analyzing nuclear activation, are also claimed as part of thepresent invention.

The nucleus of a non-dividing fetal cell or a sperm cell is normallysmall, has condensed chromatin, and does not replicate or divide.Specific nucleic acid sequences in the nucleus of these cells can bestained by fluorescent in situ hybridization methods if the targetnucleic acid sequence is accessible to the probe. However, the smallsize of the nucleus can affect the accessibility of particular nucleicacid sequences and the amount of information obtained from successfulhybridization. Moreover, hybridization signals successfully obtained arelimited in spacial resolution by the size of the nucleus. As a result,obtaining a reliable fluorescent signal can be difficult and theinformation obtained by fluorescent staining generally indicates onlythe presence or absence of accessible specific sequences, and possiblythe number of such sequences per nucleus.

In the featured methods, the present invention brings about one or morestages of nuclear activation: nuclear swelling, chromatindecondensation, DNA replication, and formation of metaphase chromosomes.Genetic information can be obtained from each of these stages, which arecharacterized by changes in nuclear structure and function. Usefulinformation obtained from these stages of activation includefacilitating the visualization of a particular chromosomal region usinga probe by increasing the spacial resolution during swelling therebyincreasing accessibility of the chromosomal region to the probe;detecting the number of a particular type of chromosome initiallypresent by determining the increased number of the particular chromosomebrought about by replication; and visualizing chromosomal morphology bystaining metaphase chromosomes, including the presence of one or moresequences at specific locations within chromosomes.

Thus, in the first aspect, the invention features a method for causing anucleus from a human fetal cell to activate. Activation is brought aboutby contacting a pretreated or, preferably, a further pretreated nucleus,with activating egg extract.

The present invention can be used to study fetal cell nuclei acidisolated by different procedures. For example, fetal cells can beobtained from circulating maternal blood, or by techniques such asamniocentesis or chorionic biopsy. Preferably, the fetal cell isobtained in a non-invasive manner (e.g., without disturbing the womb).Fetal cells such as erythrocytes and leukocytes cross the placenta andcirculate transiently in maternal blood. Furthermore, trophoblasts whichform the outermost placenta layer can pinch off and circulate inmaternal blood. Trophoblasts typically end up trapped in the maternallung capillary network.

Nuclear isolation and pretreatment is preferably carried out using mildconditions. Mild conditions are those which allows for nuclear isolationand pretreatment while causing the minimal amount of protein and nucleicacid damage. Using mild conditions helps maintain the integrity of thenucleic acid thereby decreasing artifacts during subsequent staining,and prevents premature protease activation thereby allowing subsequentprotease treatment to occur under controlled conditions chosen tooptimize such treatment.

Preferably, nuclear isolation and pretreatment to release a nucleus fromits surrounding cytoskeleton thereby forming a pretreated nucleus iscarried out in two steps; (1) membrane permeabilization, and (2)separation or alteration (e.g., denaturation or degradation) ofcytoskeletal proteins and nuclear matrix proteins. These steps may becarried out simultaneously or separately. Formation of a pretreatednucleus is preferably carried out under conditions minimizing nucleicacid damage and damage to histones.

Membrane permeabilization, opens up the membrane thereby facilitatingsubsequent nuclear treatment. Different techniques may be used formembrane permeabilization including hypotonic shock, shearing anddetergent. Preferably a non-ionic detergent is used to permeabilize theplasma and nuclear membranes. More preferably, lysolecithin is used asthe non-ionic detergent.

Different procedures can be use to separate, denature, and degrade thecytoskeletal proteins surrounding the nucleus and nuclear matrixproteins within the nucleus. These procedures include the use of a thiolreducing agent to denature nuclear protein, using controlled saltextraction to selectively remove cytoskeletal and nuclear matrixproteins, and using controlled poly-anionic treatment to facilitateseparation of negatively charged nucleic acid from the positivelycharged nuclear proteins. Separation conditions should be chosen toensure a minimal amount of damage to nucleic acids, histones, andnon-cytoskeletal proteins. Preferably, a protease is used under mildconditions to remove cytoskeletal proteins surrounding the nucleus. Morepreferably, trypsin is used as the protease. In the most preferredembodiment, pretreatment is achieved using trypsin and lysolecithin.

Activating egg extracts are used to bring about nuclear activation.Activating egg extracts contain material, such as precursors,protein(s), nuclear envelope vesicles and mRNA, which support nuclearactivation. An egg can be chemically, physically, or electricallyinduced to produce material which brings about nuclear activation. Eggscan be induced using a calcium ionophore as described below. The inducedegg continues in its cell cycle. It appears that when an egg is at thepoint in the cell cycle just prior to the S-phase, the egg cytoplasm ismost active in supporting activation. As the egg proceeds into and pastthe S-phase, it appears to produce material inhibitory to activation.

Preferably activating egg extracts are prepared from Xenopus eggs. Morepreferably activating egg extract are prepared from eggs having anelevated DNA synthesis activation activity. Activating egg extractprepared from Xenopus eggs induced for 10 minutes at 20° C. containapproximately 59% of the optimal DNA synthesis activation activity ofXenopus eggs induced for 25 minutes at 20° C. At about 25-30 minutes at20° C. the Xenopus eggs are at highest (optimal), or peak, DNA synthesisactivation activity. Xenopus eggs induced for 40 minutes at 20° C.appear to have a lower DNA synthesis activation activity than the peakactivation activity. Thus, the present invention discloses the use ofinduced eggs having an elevated DNA synthesis activation activity of 70%or greater of the peak activation activity.

Activating extracts prepared from Xenopus eggs induced for 10 minutes orless at 20° C. produce a lower rate of DNA replication in treatednuclei. However, activating extract prepared from Xenopus eggs inducedfor 10 minutes at 20° C. appear to produce equivalent or greater nuclearswelling in treated nuclei than extracts prepared from Xenopus eggsinduced for more than 10 minutes at 20° C.

More preferably activating egg extract is prepared from a number of eggs(e.g., 1,000 to 10,000), most or all of which have an elevated or peakDNA synthesis activation activity. Obtaining a large number of eggshaving a peak or elevated DNA synthesis activation activity ispreferably achieved using hardened eggs which have been synchronouslyinduced. Hardened eggs are prepared by hardening the vitelline envelopesurrounding the egg (described in detail below). Hardened eggs are lesslikely to spontaneously activate than soft non-hardened eggs.

Thus, by using hardened eggs a large number of eggs can be collected andinduced at the same time (synchronously induced). A given number of eggssynchronously induced should all be at or near the same point in theircell cycle at a given later time. A large number of eggs having anelevated DNA synthesis activation activity can be obtained by inducingthe eggs at one time, and preparing the activating egg extract from allthe eggs at a second later time. Preferably the activating egg extractis stored frozen. Freezing the extract allows a large amount of extractto be prepared at one time and used at different later times.

Various supplements to activating egg extract have been found toincrease the activation activity of the activating egg extract. Thesesupplements include cell cycle regulatory proteins, cell cycleinhibitors, cAMP (preferably, between 0.1 and 1.0 mM, most preferably at0.3 mM), and phosphodiesterase inhibitors (preferably caffeine, morepreferably caffeine at a concentration between 0.1 and 10.0 mM, mostpreferably caffeine at a concentration of 1 mM).

In another preferred embodiment activation occurs under nuclearnon-duplication conditions wherein the nucleus swells, replicates DNA,forms metaphase chromosomes and prepares to divide (i.e., entersmitosis), but segregation of sister chromatids is prevented byinhibiting spindle formation. The inhibition of spindle formationprevents the division of the cell nucleus and the resulting separationof metaphase chromosomes.

Thus, under non-duplication conditions metaphase chromosomes aredetectable for a longer time period and are provided in a “spreadpattern.” A “spread pattern” refers to the orientation of differentchromosomes with respect to each other. Drugs such as nocodazole,colchine, or colcemid can be used to inhibit spindle formation.Preferably nuclear non-duplication conditions is achieved by addingnocodazole to the activating egg extract. More preferably, nocodazole isin an amount which will not inhibit DNA replication (e.g., less than 5μg/ml).

In other preferred embodiments, prior to being treated with theactivating egg extract, the pretreated nuclei are further pretreated bycontact with a CSF extract, or a purified component of the CSF extractincluding a purified kinase or a purified phosphatase. By “purified” ismeant the component is more concentrated (e.g., has a higher specificacitivity) than when present in a CSF extract. The desired purifiedkinase or phosphatase can be obtained by purifying the enzymes from CSFfractions and assaying for activation activity. Further pretreatmentwith CSF is preferably carried out under conditions not resulting innucleus activation. Premature activation occurring under non-controlledconditions decreases the ability of CSF extracts to enhance activationbecause activation is occurring in CSF extract under non-optimizedconditions. Another disadvantage of premature activation is that itproduces a pool of nuclei activated at different times, which is moredifficult to examine than nuclei activated at the same time.

CSF extracts can be used to increase nuclear activation upon subsequentcontact with an activating agent. CSF extracts can be prepared fromnon-induced eggs (i.e., eggs arrested in meiotic metaphase II oractivated eggs that have been arrested in mitotic metaphase). Theseextracts contain factors which aid in nuclear activation, such as CSFand mitosis promoting factor (MPF). MPF may help bring about activationand visualization of chromosome by stimulating chromosome condensationand inhibiting spindle assembly.

A preferred source of CSF extracts is Xenopus eggs. Isolation of CSFextract from Xenopus eggs is facilitated using “hardened eggs” which donot spontaneously induce. Preferably, the CSF extract is stored frozen.Freezing the extract allows a large amount of extract to be prepared atone time and used at different later times.

CSF extract is preferably supplemented with reagents such as β-glycerolphosphate, creatine phosphate, phosphocreatine kinase, and Ca²⁺ inamounts which improves activation of nuclei in activating extract,without causing the start of the cell cycle prior to contact withactivation egg extract. Preferably, the CSF extract contains Ca²⁺ in anamount which leads to an increase in the level of histone kinase or MPFactivity without initiating the cell cycle. The use of Ca²⁺ tosupplement CSF extract is particular advantageous if the CSF extract isfrozen before use. The Ca²⁺ may be added before freezing or afterthawing.

Ca²⁺ is a cofactor for calmodulin activated protein kinases and mayincrease CSF activity by increasing the level of phosphorylatedtopoisomerase II activity. Topoisomerase II is a scaffold protein whichaids in chromosome decondensation and condensation possibly by anchoringchromatin loop domains. Wood and Earnshaw, J. Cell Biology 111: 2839(1990). Ca²⁺ also appears to increase the histone kinase level, which wehave used as one measure of MPF activity.

As would be appreciated by one skilled in the art, the optimal amount ofCa²⁺ added to a CSF extract varies depending upon the presence of a Ca²⁺chelator. The Ca²⁺ concentration is preferably equal to or greater than100 μM; more preferably the Ca²⁺ concentration is between 100 μM and 400μM. These preferred concentrations were determined using a CSF extractsupplemented with 1 mM ethylene glycol-bis(β-aminoethylether)N,N,N′N′-tetraacetic acid (EGTA).

In another preferred embodiment, nuclei are activated undernon-synthesis conditions which inhibit nucleic acid synthesis. As aresult, the nucleus swells with or without formation of a nuclearenvelope but does not replicate DNA or enter mitosis. The resultingincreased spacial resolution brought about by nuclear swellingfacilitates the use of nucleic acid probes by making regions of nucleicacid more accessible. Non-synthesis conditions, which neverthelesspermit nuclear swelling may be achieved by the addition of reagents suchas aphidicolin (e.g., 50-100 μg/ml), 6-dimethylaminopurine (e.g., at 5mM), leupeptin (e.g., at 5 μg/ml) dideoxycytidine triphosphate (e.g.,0.1 mM) or dideoxythymidine triphosphate (e.g., 0.1 mM) to an activatedegg extract, or to CSF extract which is then contacted with an activatedegg extract.

In another aspect, a non-dividing human nucleus is further pretreatedfor subsequent activation by contact with a purified protein kinase or apurified phosphatase which is present in a CSF extract. The purifiedprotein kinase or purified phosphatase is in a purer form (e.g., moreconcentrated or more active) than that found in a CSF extract.

In a third aspect, the invention features a method for activating anon-dividing human nucleus by further pretreating the non-dividing humannucleus in CSF extract, prepared from hardened eggs, and then contactingthese further pretreated nuclei with an activating egg extract preparedfrom synchronously induced hardened eggs.

In preferred embodiments the CSF extract is frozen before use and theactivating egg extract is frozen before use.

In other preferred embodiments, pretreated nuclei undergo furtherpretreatment in CSF extract involving a warm-then-cold incubationregime. Both the warm and cold steps increase activation of nuclei uponsubsequent contact with activating extract. Preferably, incubation iscarried out at about 25° C. for at least 30 minutes followed byincubation at about 4° C. for at least 30 minutes. Less preferred, butstill an effective incubation, is a warm regime at about 25° C. for atleast 30 minutes.

In other preferred embodiments, thawed CSF extract is supplemented withCa²⁺ in an amount which does not start the cell cycle but improvesnuclei activation. The Ca²⁺ should be in an amount which leads to anincrease in the level of histone kinase or MPF without initiating thecell cycle.

In another aspect, methods are described for preparing an activating eggextract, from hardened eggs, which can cause non-dividing human nucleicells to activate. The activating egg extracts are prepared fromhardened eggs which have been synchronously induced such that theactivating egg extract is prepared from eggs having an elevated DNAsynthesis activation activity. Preferably synchronous induction iscarried out using eukaryotic cells, more preferably amphibian, yeast,human, echinoderm, mollusc, or fish, or chicken cells are used; morepreferably Xenopus eggs are used; even more preferably Xenopus eggsinduced for more than 10 minutes are used; most preferably Xenopus eggsare induced for 25-30 minutes at 20° C.

In another aspect a method for inducing swelling in non-dividing nucleiis described. The method can be used to induce swelling in the absenceof an activating extract and in the absence of DNA synthesis. Inparticular, CSF extract is supplemented with a protein kinase inhibitorand/or an aqueous solution.

In another aspect, a method for chromosome formation without DNAreplication is described. The method involves using a CSF extractsupplemented with a cyclin such as cyclin-

90 in an amount sufficient to enhance nuclear envelope breakdown andnuclear chromosome formation. The cyclin is thought to act by raisingthe level of MPF activity in a CSF extract.

In another aspect, a method for activating a mammalian sperm cellnucleus is described. The method involves the steps of: (a) pretreatinga sperm cell, using a membrane permeabilizer, a protease, and a thiolreducing agent to form a pretreated sperm cell; and (b) activating thepretreated sperm cell. The method can be used to study sperm fromdifferent mammals. Such studies can be carried out, for example, todetermination whether the sperm contains a particular gene or nucleicacid sequence which can be passed on during fertilization.

In another aspect, activation assays are described. These assays can beused to measure different stages of activation. A basic assay comprisesisolating a nucleus, pretreating the nucleus, further pretreating thenucleus, contacting the further pretreated nucleus with an activatingegg extract and measuring activation activity. Measurement of activationactivity can be carried out using standard techniques such asincorporation of labelled nucleotides into newly synthesized nucleicacid and microscopic visualization of nuclear swelling and metaphasechromosome formation.

Other activation assays are performed by altering one or more of thesteps of the basic assay. For instance, to assay for important factorsin CSF extract, rather than using whole CSF extracts, fractions of theextract can be used. These fractions are obtained using standardpurification techniques. Similarly, different activating egg extractfractions can be studied.

In a preferred embodiment, a sperm activation assay, particularly usefulto study human male fertility, is described. Uses of the spermactivation assay include, determining the effect of handling sperm underdifferent condition thereby obtaining optimal handling condition forsubsequent in vitro fertilization, and testing the effect of possiblemale contraceptives on activation.

In other aspects viral integration assays involving the use of a cellnucleus or a pseudonucleus are described. Viral integration into a cellnucleus can be assayed as follows: pretreating a cell nucleus toseparate the nucleus from its surrounding cytoskeleton; activating thepretreated nucleus and incubating with a viral integration complexcontaining viral nucleic acid; and measuring integration of viralnucleic acid into nucleic acid of the cell nucleus. The viralintegration complex containing viral nucleic acid can be added atdifferent times during nuclear pretreatment and activation.

The viral integration assay using a pseudonucleus involves: a)constructing a pseudonucleus from a defined DNA template; b) replicatingthe pseudonucleus; and c) incubating the pseudonucleus in the presenceof a viral integration complex containing viral nucleic acid. Thisintegration complex can be added at any time during pseudonucleusformation or replication. A pseudonucleus can be constructed, forexample, by adding plasmid DNA to a CSF extract or an activatingextract. The plasmid forms chromatin in the CSF extract but does notreplicate until Ca²⁺ (1-4 mM) is added. Activation of the extractcontaining the pseudonucleus leads to nuclear envelope formation aroundthe chromatin template and causes the chromatin to replicate.

In another aspect, a product for further pretreatment of nuclei isdescribed. The further pretreatment product comprises CSF with extractsupplemented with Ca²⁺. Ca²⁺ is provided in an amount which increasesnuclei activation upon subsequent contact with activating extract.Preferably, the CSF extract is also supplemented with β-glycerolphosphate, creatine phosphate, and phosphocreatine kinase, In preferredembodiments the CSF extract is frozen; the Ca²⁺ concentration is equalto or greater than 100 μM; more preferably the Ca²⁺ concentration isbetween 100 μM and 400 μM. These preferred embodiments were determinedusing a CSF extract supplemented with 1 mM EGTA.

In another aspect, a product for causing nuclear swelling is described.The product contains CSF extract supplemented with a protein kinaseinhibitor and/or an aqueous solution.

In another aspect a product for causing chromosome formation without DNAreplication is described. The product is made up of a CSF extractsupplemented with a cyclin such as cyclin-

90 in an amount sufficient to bring about nuclear envelope breakdown andnuclear chromosome formation.

In another aspect, a product for causing a non-dividing nucleus toactivate is described. The activating product comprises an activatingegg extract prepared from an egg(s) having an elevated DNA synthesisactivation activity.

In a preferred embodiment activating egg extract is prepared fromXenopus eggs synchronously induced for more than 10 minutes; preferablythe Xenopus eggs are induced for 25 to 30 minutes at about 20° C.

In other preferred embodiments, the activating egg extract is modifiedby supplementation with cell cycle regulatory proteins, cell cycleinhibitors, cAMP (preferably, between 0.1 and 1.0 mM, most preferably at0.3 mM), and phosphodiesterase inhibitors (preferably caffeine, morepreferably caffeine at a concentration between 0.1 and 10.0 mM, mostpreferably caffeine at a concentration of 1 mM).

In another aspect, a kit is disclosed for activating a non-dividingnucleus. The kit is comprised of frozen activating egg extract preparedfrom an egg having an elevated DNA synthesis activation activity andfrozen CSF extract.

In a preferred embodiment the CSF extract contains Ca²⁺. In a mostpreferred embodiment the kit contains a microchamber microscope slide.

In another aspect, the invention features a microchamber microscopeslide provided with an upper surface having a water-repellent materialof a known thickness defining a microchamber on the upper surface. Themicrochamber is shaped to enhance flushing of the microchamber, andconnected by at least one channel to a well on the upper surface.

In preferred embodiments, the microchamber is teardrop-shaped orpear-shaped; preferably two wells are provided at opposite ends of themicrochamber connected by two separate channels to the microchamber; andthe microchamber has a defined volume preferably between 5 and 50 μl,more preferably between 10 and 20 μl when a coverslip is placed over it.Fluid can be introduced into the microchamber by placing fluid in onewell and allowing it to flow through the microchamber to the oppositewell. The fluid is then removed from the opposite well. Removal may beachieved by pipetting away the fluid or by capillary action by placementof a filter paper within the well.

In other preferred embodiments, the water-repellent material is a tapeor a coating on the upper surface of the slide, more preferably aTEFLON^(•) coating, or a wax film (e.g., a PARAFILM^(•)). In mostpreferred embodiments, the upper slide surface is treated to enhancecell growth compared to an untreated slide, the slide is provided in asterile condition, and/or the slide is coated with an antibody able tospecifically bind to a human fetal cell.

The advantages of the present invention include, but are not limited to,facilitating prenatal screening by optimizing conditions for nuclearactivation, which causes the nucleus of a fetal cell to either swell,replicate nucleic acid, and/or form metaphase chromosomes. Importantinformation regarding nucleic acid sequences or chromosome morphologycan be readily obtained from these various stages of activation, forexample by using DNA probes or visualizing the produced metaphasechromosomes. Because some fetal cells, such as trophoblasts,erythrocytes, and leukocytes can be obtained from a maternal source, anadvantage of the invention is a non-invasive procedure to detect thepresence of genetic defects in such cells.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

DRAWINGS

FIG. 1 shows the effect on DNA replication of activated nuclei, of usingfrozen/thawed activating egg extracts supplemented with caffeine.

FIG. 2 shows the effect on DNA replication of activated nuclei, of usingCSF extract supplemented with 6-dimethylamino-purine (DMAP).

FIG. 3 shows the effect of various warm-then-cold protocols on DNAreplication in activated nuclei.

FIG. 4 is a top view of a microchamber microscope slide.

METHODS AND PRODUCTS

Methods for activating nuclei include those described by Coppock et al.,Developmental Biology 131: 102 (1989); Wangh, J. Cell Science 93: 1(1989); Wood and Earnshaw, J. Cell Biology 111: 2839 (1990); Leno andLaskey, J. Cell Biology 112: 557 (1991); Young, Biology of Reproduction20: 1001 (1979); Philpott et al., Cell 65: 569 (1991); Shamu and Murray,J. Cell Biology 117: 921 (1992); Adachi et al., Cell 64: 137 (1991);Newport and Spann, Cell 48: 219 (1987); and Henry Harris, in CELL FUSION40-50 (Harvard University Press 1970).

DiBerardino et al., Proc. Natl. Acad. Sci. USA 83: 8231 (1986), and Orret al. Proc. Natl. Acad. Sci. USA 83: 1369 (1986) describe nucleartransplantation experiments to activate Rana pipiens nuclei. DiBerardinowas able to obtain tadpoles having a survival rate of up to a month, bytransplanting differentiated somatic cells into enucleated eggs.

The present invention discloses methods and products useful inactivating a non-dividing nucleus, and studying such activation. Thesemethods and products are especially useful for analyzing a nucleus fromnon-dividing human fetal cells such as aminocytes, keratinocytes,trophoblasts, erythrocytes and leukocytes. However, the methods andproducts are also useful for activating the nuclei from other types ofnon-dividing human cells such as other types of non-dividing fetal cellsand sperm, and non-dividing cells isolated from other mammals.

Preparing a nucleus for nuclear activation and bringing about nuclearactivation is described in detail below as four different phases: (1)preparation of non-dividing human nuclei, (2) preparation of activatingegg extracts from a source such as activated Xenopus eggs, (3)preparation of non-activated CSF extracts from a source such asnon-activated Xenopus eggs, and (4) activation of non-dividing humannuclei.

Also described in detail below are modified CSF extracts which can bringabout nuclear swelling in the absence of an activating egg extract; newprocedures of pretreating a sperm cell to enhance its activation; amicrochamber microscope slide which facilitates bringing about nucleiactivation and analysis of nucleic acids in such cells; a kit forbringing about nuclear activation; an activation assay; and a procedurefor cloning whole organisms from somatic cell nuclei.

The featured methods and products can be used to cause activation of anon-dividing human nucleus thereby inducing swelling, and/or DNAreplication and/or the formation of metaphase chromosomes. Theprocedures provided herein regarding nuclei activation are generallybased upon existing procedures used in other systems. However, severalimprovements over the existing systems are disclosed. Furthermore,existing procedures have not previously been used on human fetal cellsnor was it known if they would produce useful results on such cells.

Examples are given to illustrate different aspects and embodiments ofthe present invention. It is to be understood that various differentmodifications are possible and are contemplated within the true spiritand scope of the appended claims. There is no intention, therefore, oflimitations to the exact process or disclosure herein presented.

In particular, there is shown below the activation of a human fetal redblood cell nucleus using frozen/thawed activating egg extract withoutfurther pretreatment, under non-dividing conditions. The treated nucleiswelled significantly, replicated DNA, and then entered and arrested inthe pre-mitotic state. Such nuclei are useful for prenatal diagnosis.Furthermore, the use of further pretreatment, should increase the rateand extent of nuclear swelling, decrease the time it takes for DNAsynthesis to occur after activation, increase the rate and extent of DNAsynthesis, and improve the efficiency with which metaphase chromosomesare formed.

I. Nuclear Activation

(1) Preparation of Nuclei

The present invention provides a method for activation non-dividingmammalian cell nuclei, preferably non-dividing human cell nuclei. Beforebeing activated non-dividing human nuclei are isolated and pretreated. Apreferred source of non-dividing human cells are fetal cells recoveredfrom the blood of pregnant women such as trophoblasts, erythrocytes andleukocytes (such as granulocytes, neutrophils, basophiles andeosinophils). Isolating these cells does not require penetration of thewomb. The present invention is also useful for analyzing other types ofnon-dividing human cell nuclei, including non-dividing keratinocytes(e.g., those isolated from amniotic fluid), aminocytes, and sperm cells,or similar cells obtained from mammals other than humans.

Non-dividing fetal cells can be recovered from maternal blood supplyusing techniques such as antibody staining followed by cell sorting.(For example, see Bianchi entitled Non-Invasive Method For Isolation andDetection of Fetal DNA” PCT/US90/06623, hereby incorporated by referenceherein). Antibody cell sorting techniques separate fetal and maternalcells based on the presence of different antigens on fetal and maternalcells. The antigen can be differentiated by suitable antibodies. Suchantibodies which can be obtained by one skilled in art include HLe-1which recognizes an antigen present on mature human leukocytes, such asgranulocytes, and very immature erythrocyte precursor but not nucleatedcells, and antibodies to the transferrin receptor. (E.g., see Bianchi,supra PCT/US90/06623.) Procedures using antibodies can be carried out bycontacting a sample containing fetal and maternal blood with a labeledantibody recognizing either fetal cells or maternal cells. The antibodylabeled cell can be sorted using standard techniques including flowcytometry, immunomagnetic beads and cell panning.

Non-dividing human cells should be isolated under mild conditionsdesigned to prevent activation of extracellular proteases (for instancethose of the plasma), intracellular proteases, or nucleases, as well asto prevent mechanical damage to cell structures. Inadvertent protease ornuclease activation during nuclear isolation could result in damagingboth the genetic material of the cell and the protein structures withinor around the nucleic acid. Possible nucleic acid damage includes,nucleic acid degradation, and damage to the structural state (e.g.,supercoiling). One advantage of keeping the protein structure intact, ismaintaining the cytoskeletal protein so it can be subsequently separatedfrom nucleic acid under mild conditions minimizing damage to histonesand non-skeletal proteins.

Preferably, solutions used to isolate cells contain protease inhibitor.Solutions used to isolate cells such as HBSS and NIB solutions can besupplemented with protease inhibitors as follows: 0.1 mg/ml heparin, 0.1mM TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), 0.1 mM TLCK(Na-p-tosyl-L-lysine chloromethyl ketone), 0.05 mM PMSF(phenylmethylsulfonyl fluoride), 5 μg/ml leupeptin, or 31.25 mM Na₂S₂O₅.

After cell purification, the cell nucleus is preferably isolated andpretreated under mild conditions. Nuclear pretreatment is preferablycomprised of two steps, which may be carried out simultaneously orseparately; (1) membrane permeabilization, and (2) separation oralteration (e.g., denaturation and degradation) of cytoskeletal proteinsand nuclear matrix proteins. Treatment should be carried out to minimizethe damage to nucleic acid within the nucleus.

The separation or alteration of certain protein appears to be anecessary step for activation. In Xenopus erythrocytes, for instance,proteolytic digestion of cytoskeletal proteins, such as vimentin,appears to be a necessary step for subsequent nuclear activation.Coppock et al. Developmental Biology 131: 102 (1989). The pretreatmentshould prepare the nucleus for subsequent activation rather than causeactivation.

Desired conditions for plasma membrane permeabilization include milddetergent treatment, mild protease treatment, mild shearing, and mildhypotonic shock. Mild conditions are those conditions able topermeabilize the plasma membrane while creating the least amount ofdamage to the nuclear DNA and proteins. Permeabilization can be detectedusing trypan blue. Trypan blue is a dye which cannot enter intact cells.The entrance of trypan blue into a cell indicates permeabilization.Protein degradation due to inadvertent protease activation can bedetermined using polyacrylamide gel electrophoresis to look for proteindegradation products. The intactness of nuclear nucleic acids can beestablished by using agarose gel electrophoresis to determine thepresence of nucleic acid degradation products.

Possible pretreatments for separation or alteration of cytoskeletalproteins and nuclear matrix proteins include the following:

-   -   (a) Treatment with one or more thiol reducing agent such as 10        mM dithiothreitol for a limited time, at a controlled        temperature and pH, to denature cytoskeletal protein;    -   (b) Controlled salt extraction, such as by washing in buffers        supplemented with increasing amounts of NaCl or KCl in the range        of 0.025 to 1.0 M, to selectively remove cytoskeletal proteins        and proteins bound to DNA;    -   (c) Controlled poly-anion treatment, such as heparin at 0.01-1.0        mg/ml or penta sodium tripolyphosphate at 70 mM, in 10 mM borate        buffer (TPP) at pH 9.0, to selectively remove positively charged        cytoskeletal protein;    -   (d) Degradation of cytoskeletal proteins using a protease.

The extent of protein and DNA damage can be measured as described above.Preferably, nuclear isolation and pretreatment are both accomplished atthe same time using mild concentrations of lysolecithin (e.g., 40 μg/ml)and protease (e.g., 0.3 μg/ml trypsin), such that a minimal amount ofdamage to non-cytoskeletal proteins, histones, and nucleic acid occurs.The minimum time and temperature required for detergent and proteasetreatment should be used. In the case of red blood cells this is about10 minutes at 25° C., using 0.3 μg/ml of trypsin and 40 μg/ml oflysolecithin. As would be appreciated by one skill, the preferred timeand temperature will change as the concentration of the reagents change.

Controlled treatment with ion-selective chelating agents may also beperformed as an additional pretreatment. Suitable ion-selectivechelating agents include EGTA which can chelate Ca²⁺, EDTA which canchelate Ca²⁺ and Mg²⁺, and mimosine which can chelate of Cu²⁺, Al³⁺, andFe³⁺. These ions stabilize higher order chromatin structure, thus theirchelation may aid in chromatin decondensation.

Methods to terminate the detergent and protease pretreatment includeadding proteins to adsorb detergents (such as 0.4% bovine serum albumin,the bovine serum albumin employed at this step should be prepared bydialysis of commercially available BSA fraction V against distilledwater to remove soluble salts followed by lyophilization), and addingprotease inhibitors (such as soybean trypsin inhibitor) to the reaction.The pretreated nuclei should be subsequently washed using an ice coldsolution designed to preserve genomic DNA intactness. NIB buffer can beused for this purpose. NIB is made up of 250 mM sucrose, 25 mM NaCl, 10mM Pipes, 1.5 mM MgCl₂, 0.5 mM spermidine, and 0.15 mM spermine, pH 7.0.

The overall efficacy of mild conditions to obtain a pretreated nucleuscan be determined by: a) microscopic examination of nuclei to assesswhether nuclei are free of their surrounding cytoskeleton and are freestanding or clumped, clumping of nuclei is a strong indication ofnuclear damage since many nuclei get trapped in released DNA; and b) theability of nuclei to respond to activating egg extract, the use of mildconditions increases subsequent activation of individual nuclei andimproves the synchrony and homogeneity with which the entire populationof nuclei is activated.

(2) Preparation Of Activating Egg Extract

Activating egg extracts can be used to cause non-dividing nuclei toswell, assemble nuclear envelopes and lamina, replicate their genomes,enter mitosis, and form metaphase chromosomes. Activating egg extractscontain material, such as precursors, protein(s), nuclear membranevesicles, or mRNA required to activate non-dividing nuclei.

Non-activated eggs can be triggered en masse to produce material whichbrings about activation, by being chemically induced to enter the cellcycle. Eggs can be induced using standard techniques such as electricshock, pricking with a needle, fertilization and the use of a calciumionophore. (See, Gerhart, et al., J. of Cell Biology 98: 1247, 1984, andthe procedures described below.) The induced eggs enter into the cellcycle. It appears that when an egg is at the point in the cell cyclejust prior to the S-phase, the egg cytoplasm is most active insupporting activation. As the egg proceeds into and past the S-phase, itappears to produce material inhibitory to nuclear activation (see Table1).

One of the benefits of the disclosed procedures is obtaining anactivating egg extract having a higher DNA synthesis activation activitythan activating egg extract disclosed in the prior art. The DNAsynthesis activation activity can be determined by measuring thesynthesis of DNA using labelled precursors.

Hardened Xenopus eggs are a good source for preparing an activating eggextract. Hardened Xenopus eggs are stable for several hours. Incontrast, “soft” Xenopus eggs must be used rapidly. As soon as soft eggsare dejellied they tend to induce spontaneously and randomly. This isconsidered disadvantageous because activating egg extracts prepared froma specific time during the cell cycle, just prior to the S-phase, have ahigher DNA synthesis activating activity than extracts prepared fromother phases of the cell cycle. Thus, it is desirable to synchronouslyinduce a large number of eggs which are all at the same point of thecell cycle, so extracts can be prepared later from a large number ofeggs all of which have elevated DNA synthesis activation activity at thesame time.

Freshly ovulated Xenopus eggs can be hardened by stabilizing the eggsvitelline envelope as described by Wangh, J. Cell Science 93: 1 (1989).Obtaining freshly ovulated eggs from female Xenopus is facilitated byinjecting hormones which cause Xenopus to ovulate. Injecting 600 unitsof human chorionic gonadotropin (HCG) into a Xenopus female generallybrings about ovulation within 12-15 hours. Injection of pregnant mareserum gonadotropin about 24 hours before HCG treatment significantlyincreases the yield of mature eggs. Furthermore, repeated ovulation offrogs once every 4-8 months improves the yield of eggs by increasing thesynchrony of oocyte development in the ovary.

The freshly ovulated eggs within their jelly coat, are flooded with0.3×NKH (1×NKH is 40 mM NaCl, 2.5 mM KCl, 7.5 mM Hepes, pH 7.4 withNaOH), for 15-20 minutes. During this time the jelly layers swell. Theeggs are then dejellied in 3×NKH containing 2% cysteine, pH 7.9, bygentle swirling for about 5 minutes.

The resulting soft eggs can be “hardened” by immediately rinsing themfive times in 3×NKH containing 2 mM MgCl₂, 1 mM CaNO₃, 10 μM ZnCl₂ andletting them stand for at least 20 minutes at room temperature. Hardenedeggs are sorted to remove damaged and partially induced eggs. Calcium isrequired for hardening and must be present in the 3×NKH used to washcysteine-treated eggs. Eggs washed in the absence of Ca²⁺ andsubsequently treated with Ca²⁺, and Ca²⁺ treatment of eggs still in thejelly coat, do not result in hardened eggs. Additionally, adding Ca²⁺before or during dejellying will not result in hardening.

Activating egg extract is preferably obtained from hardened eggs induceden masse. Induction can be carried according to procedures described byCoppock et al., Developmental Biology 131: 102 (1989). The proceduredescribed by Coppock et al. as modified, in the following manner, wasused to obtain “prepared activating egg extract”: 5-15 ml of hardenedeggs were rinsed using activation buffer (4 mM NaCl, 0.14 mM potassiumgluconate, 2 mM Hepes, 2 mM MgSO₄, and 0.6 mM Ca(NO₃)₂, pH 7.8), andplaced in 500 ml of activation buffer; the eggs were then induced byadding calcium ionophore A23187 (10 μM in DMSO; Sigma Chemical Co.) to afinal concentration of 100 nM and incubating at room temperature; after10 minutes calcium ionophore treated eggs were rinsed and induced for anadditional 15-20 minutes by incubating in 1.5×NKH containing 2 mM MgCl₂,and 0.6 mM CaCl₂ (Coppock et al., supra, stops the induction at 10minutes); the eggs were then placed on ice in a siliconized or teflonbeaker and washed 3-5× in several hundred milliliters of ice cold EBbuffer (EB=50 mM potassium gluconate, 250 mM sucrose, 10 mM potassiumHEPES, 1.5 mM MgCl₂, pH adjusted to 7.5 with potassium hydroxide); eggswere then transferred to a volumetric polyallomer centrifuge tube, mixedwith Versilube F-50 oil (General Electric) at 0.2 ml oil/ml eggs, andtight packed by centrifugation at 40×g for 1 minute, at 2-4° C.; theoverlaying oil and aqueous layers were removed and the eggs were crushedby centrifugation 15 minutes at 9,000×g, at 2-4° C.; the cytoplasmiclayer between the yolk pellet and the overlaying lipid layer wascollected from the bottom by puncturing the tube with a syringe needle;cytochalasin B was added to a final concentration of 10-50 μg/ml and thecytoplasmic material recentrifuged for 15 minutes at 9,000×g, at 2-4°C.; the resulting second cytoplasmic supernatant was recovered andeither used fresh or frozen for future use.

This procedure for “prepared activating egg extract” involving anincreased induction time of 15-20 minutes over that described in Coppocket al. supra, was chosen based upon the following two experiments: 1)plasmid DNA injected into non-activated Xenopus eggs does not beginreplicating until 25-30 minutes after eggs are induced, during this lagperiod factors required for DNA synthesis are possibly released,altered, or synthesized within the egg; and 2) extracts prepared fromeggs induced for only 10 minutes synthesize additional proteins in vitrowhich first act to increase DNA synthesis in pretreated Xenopuserythrocyte nuclei and then act to inhibit DNA synthesis in these samenuclei.

The second experiment, “induction optimization,” is particularly usefulin determining the optimal induction time for obtaining activating eggextract having an elevated DNA synthesis activation activity. Theexperimental results for induction optimization used to obtain anactivating egg extract with an elevated DNA synthesis activationactivity from Xenopus, is shown in Table 1. The same experimental designcould be used to establish the induction time needed to obtain eggextracts having elevated DNA synthesis activation activity from speciesother than Xenopus.

Induction optimization was carried for Xenopus as describe below (seealso Example 2, infra, for further optimization experiments). Xenopuserythrocyte nuclei were isolated and pretreated with lysolecithin andtrypsin as described in Example 2 (described below). An activating eggextract was prepared from hardened eggs which were induced for 10minutes as described above. Both the activating egg extract and thepretreated nuclei were kept on ice (about 4° C.). The activating eggextract was supplemented with 1 mM ATP (not used in the other examplesdescribed herein), 10 μg/ml creatine phosphokinase, 10 mM creatinephosphate, 10 μCi P³²-dCTP and combined with pretreated nuclei (about200 nuclei/μl) Individual aliquots containing activated nuclei wereshifted from 4° C. to 25° C. Cycloheximide to a concentration of 100μg/ml was added to the individual aliquots at different times. Thealiquots were then incubated at 25° C. for a total time, including thetime at 25° C. before addition of cycloheximide, of 60 minutes. After 60minutes, P³²-dCTP incorporation into newly synthesized DNA wasdetermined. TABLE 1 Time CHM Added (Minutes) Cpm Incorporated into DNANo CHM 1,296  0 2,894  5 4,208 15 4,937 30 3,775 45 2,314

The result of induction optimization for Xenopus activating egg extractis shown in Table 1. The highest observed DNA synthesis activationactivity was 15 minutes after the addition of cycloheximide. Thus, aboutfifteen minutes appears to be the additional time required for peak DNAsynthesis activation activity (total induction time of about 25minutes). A more precise time point for the activation peak may bereadily determined by taking additional experimental time points.Elevated DNA synthesis activation activity (more DNA synthesisactivation activity than zero time), was seen after an additional 5, 15,and 30 minutes. The elevated DNA synthesis activation activity decreasedfrom 15 to 30 minute time points. After the 45 minute time point theobserved DNA synthesis activation activity was below that of theelevated DNA synthesis activation activity. The decrease in DNAsynthesis activation activity observed for incubation times longer thanan addition 15 minutes is attributed to the synthesis of proteinsinhibitory to activation.

As would be appreciated by one skilled in the art, the optimal DNAsynthesis activation time will also vary as the temperature changes. Asthe temperature increases the optimal DNA synthesis activation timedecreases, however, the temperature is preferably not raised above 24°C. As the temperature decreases the optimal DNA synthesis activationtime increases, however, the temperature is preferably not lowered below16° C.

Several proteins present in Xenopus egg extracts are involved in DNAreplication. One or more of these could be a positive acting proteinsynthesized during the first 25-30 minutes after activation responsiblefor the increase in DNA synthesis activation activity. Possible positiveacting proteins include: cyclin A, RFA single strand binding protein,cdk2 kinase, and RCC1 protein. There are also several proteins whosesynthesis after the first 25 minutes could be responsible for thedecrease in DNA synthesis activation activity. Possible proteins whichcould decrease DNA synthesis activation activity include cdc2 and cyclinB. Given the evolutionary conserved nature of both the positive andnegative acting proteins, and their functions, it is likely that eggsfrom species other than Xenopus also display an optimal time just beforethe start of S-phase when their cytoplasm is most active in supportingDNA synthesis activation.

As seen in Table 1, incubating for 10 minutes provided less than 60% ofthe peak DNA synthesis activity observed compared to the optimal DNAsynthesis activity of extracts prepared from Xenopus eggs. Using thetechniques described herein, the induction time required for obtainingegg extract having an elevated DNA synthesis activation activity (morethan 70% of the peak activation) can be obtained for activating eggextract prepared from egg sources other than Xenopus.

Activating egg extracts from “hardened” eggs may be used fresh in whichcase they support more than one cell cycle in vitro. Alternately, theseextracts may be frozen and then thawed, in which case they are able tosupport one or more cell cycles in vitro.

The activating egg extract is preferably made 7.5-10% (v/v) in glyceroland stored frozen in liquid nitrogen, by standard techniques or by anincreased rapid freezing technique. The increased rapid freezingtechnique freezes the extract faster than merely suspending in liquidnitrogen. Increased rapid freezing can be achieved by spotting extract,made 7.5-10% (v/v) glycerol, as 20 μl droplets onto a block of aluminumimmersed in liquid nitrogen.

Before use, frozen activating egg extracts are thawed rapidly at roomtemperature, put on ice, and if desired, supplemented to enhanceactivation activity. One possible supplement is cyclic-AMP. The additionof 0.1 mM to 10 mM cAMP to activating egg extracts increases theactivation activity of the activating egg extract, as measured bysubsequent DNA replication in pretreated Xenopus erythrocyte nuclei.cAMP can be broken down by phosphodiesterase. Caffeine is an inhibitorof phosphodiesterase and, thus, enhances the stability of endogenous andadded cAMP. Thus, caffeine and phosphodiesterase inhibitors are possiblesupplements to enhance activation activity of activating egg extract.Indeed, the addition of caffeine to activating egg extract was found toincrease subsequent DNA replication in activated Xenopus nuclei.

Appropriate egg extracts can be obtained from sources other thanXenopus. Useful guidelines for choosing an appropriate egg source tomake either activating egg extract or CSF extract are provided below.These guidelines are not intended to be a list of requiredcharacteristics, but rather a list of considerations useful for choosingan egg source.

Useful guidelines for choosing an appropriate egg source for making eggextracts include the following:

1. Egg/embryo with substantial stores of activating cell cycle materialare preferred. Such egg/embryos can be identified as those showing aseries of rapid cell cycles, i.e., cell divisions approximately onceevery hour as compared to once every day.

2. Moderate egg size is preferred. Moderate egg size represents acompromise between the cytoplasmic volume per egg and yolk mass per egg.Preferably a large yield of cytoplasm per volumetric measure of eggs isobtained.

3. A species in which female animals shed a large number of eggs ispreferred as a means of increasing the amount of egg extract availablefrom an animal, while keeping the cost of caring for the animal at aminimum. However in some instance, such as the activation of mammaliansomatic cell nuclei prior to transplantation into their correspondingeggs, it may be desirable to prepare extracts from mammalian eggsdespite their small size and relatively small number per female.

4. Females of the chosen species are preferably identifiable by externalcharacteristics.

5. Females preferably breed in a reasonable period of time (at leastonce per year), and at a reasonable cost.

6. Eggs are preferably shed as single cells (e.g., not in jelly mass),or easily freed of jelly layers and other major external envelopes.

7. Eggs can preferably be stabilized from activating once freed ofextracellular coats. (see e.g., Wangh, J. Cell Science 93: 1 (1989)).

8. Females preferably produce high quality eggs which are uniform andregular. These features minimize waste and help in developing automatedmethods to sort good and bad eggs. Some eggs, such as those ofechinoderms and mollusks, are transparent and contain a prominentgerminal vesicle nucleus which can be used to judge egg quality. Othereggs, such as those of Xenopus, are not transparent, but have twodistinct colors which can also be used to judge egg quality.

9. Eggs are preferably chemically inducible in a synchronous manner suchthat a number of eggs may be induced at the same time and beapproximately at the same point in the cycle at a specified later time(preferably at meiotic metaphase state or mitotic metaphase state). Inthis way, extracts may be obtained from a number of eggs at the samepoint in the cell cycle by inducing all the eggs at one time and usingall the eggs to prepare an extract at a later time.

10. Females can preferably be chemically induced to ovulate therebymaking it possible to increase the production of eggs from a givenfemale.

11. Female are preferably not harmed by the egg collection method.Alternately, if egg collection does harm the female those females forwhich a commercial use of the carcass exists are preferred.

12. Preferably the eggs allow preparation of extracts that inducenuclear swelling, either without or with concomitant DNA replication.Nuclear swelling without replication can be achieved by removal ofmembrane vesicles required for nuclear envelope assembly, or byinhibition of DNA synthesis (e.g., using reagents such as aphidicolin,mimosine, or DMAP), or by CSF extract supplemented with a kinaseinhibitor (e.g., such as DMAP or staurosporine). Nuclear swelling withreplication can be achieved using activating egg extracts such as thoseobtained from Xenopus eggs.

13. It is important that eggs used to prepare CSF extracts can bearrested in either the meiotic metaphase state, or in the mitoticmetaphase state. Recovery of chromosomes, rather than interphase nuclei,requires cell cycle arrest in metaphase. For some species, extracts inmetaphase arrest can be prepared directly from non-activated eggs, suchas unfertilized Xenopus eggs, or can be made to cycle into and arrest inmeiotic metaphase. Useful reagents for bringing about and causing arrestin meiotic metaphase include cyclin

90 (a non-degradable form of sea urchin cyclin), other cyclin relatedpeptides, small amounts of CSF extract (prepared from non-activatedXenopus eggs), components found in non-activated Xenopus eggs (such asc-MOS kinase) or Calyculin A used on echinoderm eggs (Tosuji et. al.Proc. Natl. Acad. Sci. 89: 10613 (1992)).

(3) CSF Extract Treatment of Nuclei

Non-activated CSF extract can be used to aid subsequent nuclearactivation of non-dividing mammalian cell nuclei, including human cellnuclei, without directly causing nuclear swelling or DNA replication; orto directly cause nuclear swelling as discussed in section II infra.Nuclei in CSF extract appear to condense into chromosome like structuresand may become surrounded by a spindle apparatus. Nuclear activationprior to contact with an activating egg extract is disadvantageous.Problems with premature activation include a decrease in the enhancementof activation and different nuclei being activated at different times.

The ability of CSF extract to enhance activation may be increased byvarious supplement. In addition, the incubation conditions of nuclei inCSF extract can be adjusted to improve the ability of such extracts toenhance activation of the nuclei upon subsequent contact with activatingegg extract.

The CSF extract is preferably prepared from non-induced eggs arrested atmeiotic metaphase. CSF extract prepared from non-induced eggs arrestedat meiotic metaphase contain high levels of mitosis promoting factor(MPF) activity and cytostatic factor (CSF) activity. CSF and MPF arefactors present in CSF extract which are believed to aid in subsequentactivation of quiescent nuclei by altering cytosketal proteins, nuclearmatrix proteins, and nuclear histones, particularly by phosphorylationof these proteins.

MPF is an activity controlling nuclear entry into mitosis and initiationof spindle assembly. MPF is composed of two catalytic subunits,p34^(cdc2) and cyclin B. At the onset of anaphase, cyclin B is destroyedresulting in the inactivation of MPF. During anaphase the chromosomesmove towards the two opposite poles of the spindle apparatus andsubsequently decondense.

CSF is an activity responsible for metaphase arrest in unfertilizedvertebrate eggs. CSF activity is due to at least two kinases:mitogen-activated kinase (MAP) and cdk2/cyclin (cdk2 is a kinase relatedto cdc2, but the regulatory subunit of cdk2 is cyclin E (or A) ratherthan cyclin B). The activities of MAP appears to be controlled byadditional kinases such as c-Mos kinase.

One reason for obtaining CSF extract from eggs arrested at meioticmetaphase, is that both MPF and CSF are inactivated upon initiation ofthe cell cycle.

CSF extracts from non-induced Xenopus eggs can be prepared by a methodbased on the work of Lohka and Masui, Developmental Biology 103: 434(1984), as well as that of Murray et al., Nature 339: 280 (1989). Aprocedure for obtaining CSF extract is as follows. Eggs are obtainedfrom one or more ovulating frogs as described above. Each batch offreshly ovulated eggs, about 500 to 1000 eggs, is hardened as describedabove. Damaged and activated eggs are removed. The remaining eggs arecombined into a large siliconized glass or teflon beaker and washed 4-5times at room temperature (about 21° C.) in approximately 500 mlEB-buffer containing 5 mM potassium EGTA, pH 7.5, (EB=50 mM potassiumgluconate, 250 mM sucrose, 10 mM potassium HEPES, 1.5 mM MgCl₂, pHadjusted to 7.5 with potassium hydroxide). The eggs are then transferredto a volumetric polyallomer centrifuge tube, mixed with Versilube F-50oil (General Electric) at 0.2 ml oil/ml eggs, and are tight packed bycentrifugation at 40×g for 1 minute, at room temperature. The overlayingoil and aqueous layers are removed and the eggs are crushed bycentrifugation for 15 minutes at 9,000×g, at 2-4° C. The cytoplasmiclayer between the yolk pellet and the overlaying lipid layer iscollected from the bottom by puncturing the centrifuge tube with asyringe needle. Cytochalasin B is added to a final concentration of10-50 μg/ml and potassium EGTA is added to a final concentration of 1mM. The cytoplasmic material is mixed by gently pipetting or rockingback and forth, the cytoplasmic material is then centrifuged for 15minutes at 9,000×g, at 2-4° C. An alternative centrifugation procedureinvolves preparation of a high speed supernatant by centrifugationat >100,000×g for 2 hrs at 2-4° C. In either case, the resulting secondcytoplasmic supernatant (hereinafter “prepared CSF extract”) isrecovered and is either used fresh or is made 7.5-10% in glycerol andfrozen for future use in the same manner as activating egg extract.Preferably, CSF extract is incubated at 250 C for 2 hours prior tofreezing. The level of histone H1 kinase activity increases several foldduring the period of incubation.

Frozen extracts can be used by thawing rapidly at room temperature andthen placing on ice. Thawed extracts are preferably supplemented with anATP regenerating system consisting of 10 mM creatine phosphate, and 10μg/ml creatine phosphokinase.

The histone H1 kinase activity, the structural state of plasmid DNAadded to the CSF extract, and the inability of CSF extract to causenuclear activation, demonstrated that “prepared CSF extract” wasarrested in meiotic metaphase. The histone H1 kinase activity of the CSFextract either before or after freezing was high. Upon activation of theextract with 1.2 to 4 mM Ca²⁺, the histone activity decreased.Preferably, 1.2 mM Ca²⁺ is sed when CSF extract is supplemented with 1mM EGTA to achieve recycling. No recycling occurs when 3 to 4 mM of Ca²⁺is used in CSF extract supplemented in the presence of 1 mM EGTA.Negatively supercoiled circular plasmid DNA added to the extractrelaxed. Lysolecithin-trypsin pretreated Xenopus erythrocyte nucleiadded to CSF extract failed to swell or synthesize DNA.

After further pretreatment in CSF extract, the nuclei may be activatedby adding 9 volumes of “prepared activating egg extract.” DNAreplication, measured by incorporation of labelled nucleotides into DNAstrands, may be used to determine the extent to which prior treatment inCSF extract enhances nuclear activation in activating extract. Labellednucleotides useful in measuring nuclear DNA replication includemicrocurie amounts of P³²-dCTP for radioactive measurement of newlysynthesized DNA, 16-50 μM biotinylated-dUTP or BrdUTP for fluorescentmeasurement of newly synthesized DNA, and 250 μM BrdUTP for densitylabelling of newly synthesized DNA.

Several supplements to CSF extract, in the proper concentration,increased the ability of CSF extract to enhance activation activitywithout resulting in premature DNA synthesis activity. Usefulsupplements include β-glycerol-PO₄, Ca²⁺, and protein kinase inhibitors.The addition of β-glycerol-PO₄ increased the rate at which negativelysupercoiled DNA relaxed in CSF extract and was subsequently assembledinto chromatin. A concentration of about 80 mM β-glycerol-PO₄ was foundto aid in chromatin assembly without causing DNA synthesis.Beta-glycerol-PO₄ is an inhibitor of phosphatase activity and may act byincreasing the level of the phosphorylated functionally-active form oftopoisomerase II in the CSF extract.

Similarly, the addition of 100 μM Ca²⁺ increased both the ratenegatively supercoiled DNA relaxed in CSF extract and rate of subsequentassembly into chromatin. Calcium is a cofactor for calcium calmodulinactivated protein kinases and may also act by increasing the level ofphosphorylated active topoisomerase II activity in the CSF extract. Theaddition of 100 μM CaCl₂ to thawed CSF extract failed to trigger itsentry into the cell cycle as judged by continued high levels of histoneH1 kinase activity. The addition of 100 μM Ca²⁺ also increased both theamount and the rate of DNA synthesis in erythrocyte nuclei afteraddition of activating egg extract. CSF extract responded to theaddition of 1.2-4 mM Ca²⁺ by increasing the rate and extent of chromatinassembly over that seen upon addition of 100 μM Ca²⁺. However, thehigher concentration of calcium also activated the CSF extract.

The association of factors, whose presence increase DNA synthesisactivity of CSF extract was also examined. Apparently, one or morefactors in CSF extract which aid in subsequent DNA replication areloosely held by the nuclei in CSF extract and are lost during washing.Xenopus cell nuclei were pretreated with trypsin and lysolecithin, addedto CSF extracts to a concentration of 1000-2000 nuclei per μl, andeither washed by diluting into excess NIB buffer and centrifuging, ornot washed. Subsequent addition of activating egg extract, to aconcentration of 100-200 nuclei per μl, resulted in less DNA replicationfor washed nuclei. For this reason, CSF extracted treated human nucleiare preferably not washed prior to contact with activating egg extract.

Nuclear activation upon contact with activating egg extract can beincreased by manipulating the conditions in which nuclei are incubatedin CSF extract during further pretreatment. Useful manipulations can beobtained by regulating the incubation period and temperature. The use ofa warm-then-cold regime stimulates subsequent nuclei activation. Bothwarm and cold steps appear to exert positive effects on subsequentnuclei activation. Preferably, the warm-then-cold regime comprisesincubation at about 25° C. for 30-90 minutes followed by incubation at4° C. for 30-90 minutes.

Trypsin and lysolecithin treated Xenopus red blood cell nuclei incubatedin frozen/thawed CSF extract using a warm-then-cold regime and contactedwith fresh activating egg extract resulted in extensive and synchronousnuclear envelope formation, swelling, and replication upon contact withfreshly prepared activating egg extract. While this system is attractiveto aid in activation of human nuclei, because of the convenience ofusing frozen CSF extract, use of this system on Xenopus erythrocytenuclei revealed several limitations. One limitation is the need tofreshly prepare activating egg extract, which is experimentallyinconvenient.

Using CaCl₂ in conjunction with frozen CSF overcomes this limitation.The use of CaCl₂ permits synchronous nuclear envelope formation,swelling, replication, entry into mitosis (including formation ofchromosome-like structures without DNA fragmentation), and renewed DNAsynthesis in a second S-phase when both frozen CSF and frozen activatingegg extracts are used. Thus, the use of both a warm-then-cold regime andCaCl₂ is particularly advantageous when frozen activating egg extractsand frozen CSF extracts are used to cause nucleus activation. Preferablythe CSF extract contains 0.1 to 0.4 mM CaCl₂ to enhance nuclearactivation upon subsequent contact with activating egg extract. At thisrange of Ca²⁺, nuclei treated in CSF extract should not activate untilcontact with activating extract.

(4) Activation of Nuclei with Activating Egg Extract

Activating egg extracts can be used to activate non-dividing mammaliancell nuclei, such as non-dividing human cell nuclei, to bring aboutswelling, chromatin decondensation, DNA replication and formation ofmetaphase chromosomes. However, nuclear activation can be stopped atvarious points and information about nucleic acid sequence and structurecan be obtained by examining the resulting DNA. Under duplicationconditions, the nucleus swells, DNA replicates, and the resultantchromosomes divide. Under non-duplication conditions, the nucleusswells, DNA is replicated, but the resultant chromosomes do not divide.Under non-synthesis conditions the nucleus swells, but DNA is notreplicated, and nuclei do not divide.

The use of nocodazole, or other drugs like colchine, colcemid, and D₂Owhich inhibit microtubule assembly is preferred for preventingseparation of mitotic chromosomes. These drugs prevent the formation ofmitotic spindles during the cell cycle. As a result, condensedchromosomes accumulate rather then separate to the cells poles and arereadily visualized for karyotypic analysis.

However, the addition of 5 μg/ml nocodazole to activating egg extractdecreases the rate of DNA replication. Thus, to maintain a high rate ofDNA replication it is necessary to either: 1) use nocodazole at a doseless than 5 μg/ml, such as adding nocodazole to CSF extract at 5 μg/mland diluting the mixture with 9 volumes of activating egg extract; 2)use another drug such as colchine, colcemid or D₂O (deuterium oxide)which may be able to block mitotic spindle formation without inhibitingDNA replication; or 3) add the spindle inhibitor later, i.e., after DNAsynthesis is complete but before nuclei proceed into mitosis.

To avoid artifacts such as chromosome fragmentation during nuclearactivation it is desirable that complete, rather than partial,replication of nuclear genomes be achieved. The following techniques areuseful to assess the extent of genome replication achieved duringnuclear activation:

1) Coordinate observations of the kinetics of DNA synthesis, the size ofthe DNA molecules made, the timing of mitosis following DNA synthesis,and the morphological appearance of nuclei. Complete replication ischaracterized by a early onset and rapid rate of DNA synthesis in allnuclei, an abrupt cessation of DNA synthesis in all nuclei, followed byrapid entry into mitosis, and renewed replication when nuclei exitmitosis. In addition, newly synthesized DNA molecules are very long(greater than 50,000 base pairs), but are transiently cleaved by type IItopoisomerase during the period of chromosome condensation anddecondensation.

2) The isotope dilution technique can be used to measure the pool sizeof DNA precursors in the activating egg extract to establish the extentof genome replication, on the basis of the radioactive specific activityof the DNA. The isotope dilution technique can be carried out accordingto Blow and Laskey, Cell 47: 577 (1986).

3) BrdUTP density labelling of newly replicating DNA followed byisopycnic centrifugation in CsCl and Southern hybridization can be usedto determine if one or more rounds of replication is occurring. Duringthe initial round of semi-conservative replication, incorporation ofBrUTP leads to formation of a DNA duplex having one heavy (BrUTPcontaining) strand and one light strand. The subsequent production of aDNA duplex containing two heavy strands indicates more than one round ofreplication.

4) DNA replication can be visually measured usingbiotinylated-deoxynucleotide triphosphates (such as biotin-11-dUTP) orbromodeoxy-UTP. These labeled nucleotides can be added to activating eggextracts and are incorporated into DNA during replication. Nucleicontaining the labelled DNA can be recovered and examined in afluorescent microscope. The labelled DNA is conveniently visualized bystaining with Texas Red streptavidin (for biotin samples) or FITC(fluorescein) anti-BrdUTP antibodies. Total DNA can be visualized usinga fluorescent intercalating dye (such as propidium iodide or Hoechststain) or a fluorescently tagged reagent. In some cases it may bedesirable to treat nuclei in the microchamber microscope slide with highsalt solutions to stretch the DNA across the glass surface before DNAstaining. A fluorescent microscope can be employed to establish whetherall regions of the nuclear DNA (stained for instance with Hoechst)contain newly synthesized DNA (stained for instance with biotin-TexasRed streptavidin).

II. Use of a CSF Extract to Cause Nuclear Swelling

Nuclear swelling can be brought about in non-dividing nuclei by using amodified CSF extract, made by high or low speed centrifugation, or byusing a CSF extract or modified CSF extract made by high speedcentrifugation (a “partially purified CSF extract”). Use of a CSFextract to induce nuclear swelling is preferably carried out on isolatednuclei pretreated to separate the nuclei from its surroundingcytoskeleton, preferably with detergent and a protease as describedherein. Preferably, the modified CSF extract is a partially purified CSFextract and is modified by either (a) diluting with an aqueous solutionand/or (b) supplementing with a protein kinase inhibitor. Fresh CSFextract or frozen thawed extract can be modified.

Dilution of CSF extract to enhance its ability to cause nuclear swellingmay be carried out using various aqueous solutions such as water andphysiological pH buffers. The aqueous solution is preferably buffered toabout pH 6.5 to about pH 7.5. An example of an appropriate buffer is EBbuffer (EB=50 mM potassium gluconate, 250 mM sucrose, 10 mM potassiumHEPES, 1.5 mM MgCl₂, pH adjusted to 7.5 with potassium hydroxide).Preferably, the aqueous solution is added in an amount to achieve 25% to75% dilution.

The ability of CSF extract to cause nuclear swelling can also beenhanced by using a protein kinase inhibitor such as DMAP orstaurosporine. DMAP and staurosporine are broad range kinase inhibitorsable to inhibit the actions of both CSF and MPF. Other protein kinaseinhibitor able to inhibit CSF and/or MPF can be obtained by one skilledin the art. The chosen kinase inhibitor can preferably inhibit both CSFand MPF.

Preferably, 2.5-5 mM of DMAP is used. The use of protein kinaseinhibitors should block kinase activities in the extract, includinghistone H1 kinase and result in the treated nuclei forming envelopes butfailing to initiate DNA replication. The proper protein kinase inhibitorconcentration can be empirically determined by one skilled in the art bymeasuring the extent of swelling and DNA replication in the presence ofdifferent amount of protein kinase inhibitors.

Preferably, an aqueous solution and a protein kinase inhibitor are bothused to modify CSF extract.

Nuclei treated with diluted CSF extract supplemented with DMAP(CSF-DMAP) swell to a greater volume than nuclei treated with undilutedCSF-DMAP. Preferably, CSF containing a protein kinase inhibitor(CSF-PKH) is diluted 25% to 75% using an appropriate buffer and nucleiare incubated for more than 60 minutes at 25° C., and more preferablyaround 90 minutes at 25° C. prior to measuring nuclei swelling.

Diluted CSF-PKH extract can be further modified by altering the Ca²⁺ andMg²⁺ ion concentration to further increase swelling, and affectchromatin condensation and decondensation. Ca²⁺ and Mg²⁺ ionconcentration can be altered by addition of these ions or by removal ofthese ions by using chelating agents, such as ethylenediaminetetraacetic acid (EDTA) (e.g., 5 mM), or ethyleneglycol-bis(B-aminoethyl ether)N,N,N′N′-Tetraacetic Acid (EGTA) (e.g., 5mM). Altering the free Ca²⁺ and Mg²⁺ ion concentration in the dilutedCSF-PKH has the effect of changing the extent of nuclear swelling andthe appearance of the chromatin within the nucleus. Very low or absentCa²⁺ and Mg²⁺ ion levels enhance nuclear swelling and chromatindecompaction. Increasing Ca²⁺ to 1.2 mM prevents significant nuclearswelling and chromatin decompaction. The optimal amount of Ca²⁺ andMg²⁺, and chelator can be empirically determined by varying the amountof chelator and cation concentration and measuring nuclei swelling.

Chelating agents or other agents which cause chromatin decondensation,such as polyanions like heparin and TPP or thiol reducing agents likeDTT, need not be added directly to the diluted CSF-PKH extract. Theseadditional agents may be used to further swell or decondense the nucleiafter treatment in diluted CSF-PKH extract.

III. Use of a CSF Extract Supplemented with a Cyclin

A CSF extract supplemented with a cyclin can be used to inducechromosome formation without DNA replication. The CSF extract should besupplemented with a cyclin such as cyclin-

90 in an amount sufficient to achieve both nuclear envelope breakdownand nuclear chromosome formation. The cyclin is expected to act byraising the level of MPF activity in CSF extracts, and thereby, induceconversion of isolated nuclei into mitotic chromosomes.

CSF extract supplemented with cyclin was used to activate nuclei insuspension resulting in nuclei conversion into chromosomes. However, theactivation also resulted in intermingling of the formed chromosome, andthe chromosomes were stretched and sheared. The use of CSF extractsupplemented with cyclin-

90 may be improved by sticking trypsin treated nuclei to a glass surfaceprior to incubating with modified CSF.

IV. Activation of Mammalian Sperm

The present invention also features a method for activating mammaliansperm, which is particularly suitable for the activation of human sperm.The method involves the pretreatment of human sperm with a protease,then activating the sperm with an activating egg extract. The presentdisclosure is believed to be the first describing the use of trypsin andan activating egg extract to activate a human sperm. The pretreatedsperm can be activated using activating egg extract, or the variousprocedures described herein, such as using a modified CSF extract (e.g.,supplemented with an aqueous solution and/or a protein kinase inhibitor,or supplemented with a cyclin) to achieve nuclear swelling or chromosomeformation, and using both a CSF extract and activating extract to causenuclear activation.

The preferred method for activating a sperm involves (1) pretreatmentinvolving a protease, a detergent, a thiol reducing agent, andpreferably a thiol blocking to prevent reassociation of sulfhydrylgroups; (2) further pretreatment using CSF extract; and (3) activationusing an activation extract. Sperm can be obtained using techniquesknown in the art. Pretreatment can be carried out using a protease and adetergent either sequentially, or at the same time, followed by a thiolreducing agent, followed by a thiol blocking agent.

An example of a preferred protocol is as follows:

-   -   1. Lyse sperm in 100 μg/ml lysolecithin for 5 min at 25° C.    -   2. Treat with 100 μg/ml trypsin for 5-15 minutes (10 minutes is        optimum) at 25° C.    -   3. Stop the lysolecithin and trypsin treatment by using 30 μg/ml        soybean trypsin inhibitor and 0.4% bovine serum albumin.    -   4. Incubate sperm nuclei in 5 mM dithiothreitol for 60 minutes        at 4° C.    -   5. Stop reaction 4 by incubating nuclei in 1 mM N-ethylmaleimide        for 10 minutes at 25° C.    -   6. Incubate nuclei in CSF extract for 90 min at 2-5° C.,        followed by 60 minutes at 4° C.    -   7. Incubate nuclei in activating egg extract.

The above procedure results in nuclear swelling without nuclear envelopeformation during the CSF pretreatment step (step 6), and additionalswelling, nuclear envelope formation, and DNA replication during theactivating egg extract step (step 7). Steps 1-3, or their equivalent,are all required to achieve complete swelling, nuclear envelopeformation, and DNA replication.

Activation of sperm cells have various uses including being used todetermine whether the sperm contains a particular gene or nucleic acidsequence which can be passed on during fertilization. Such studies areuseful, for example, to study the effect of aging on sperm; detectchromosomal defects; and determine whether foreign genes (such as thosepresent in the human immuno deficiency virus (HIV)) are present issperm.

An example of the usefulness of this aspect of the invention is in thefield of animal breeding, particularly the breeding of transgenicallymodified animals. Transgenically modified animals are usually created byinjecting DNA sequences into early embryos. If the injected DNAintegrates into the host cell genome, it may end up in the germ line ofthe adult animal after the animal matures. Transgenic male animals areparticularly desirable since they can be bred to many females. However,prior to breeding the percentage of modified germ cells, as well as thecopy number and distribution of the inserted genes in each cell is notknown.

The methods and reagents provided herein for activating sperm cellnuclei and examining their genetic composition, for example via in situhybridization, make it possible to determine the percentage of spermcarrying one or more copies of the inserted gene. This information canused to access the likelihood that a particular gene will be passed onto a future generation, prior to breeding the animal. Such informationis desirable because of the time and expense required to breed ananimal.

V. Nuclear Activation Assay

The procedures disclosed by the present invention, to activate nuclei,can also be used as a general assay procedure to measure nuclearactivation and the presence of a nucleic acid sequence in activatednuclei. The assay would be particularly useful to identify and purifyfactors present in CSF extract and to study male fertility.

A basic assay to measure DNA replication of activated cell could havethe following steps: isolating a nucleus, pretreating the nucleus,further pretreating the nucleus, contacting the further pretreatednucleus with activating egg extract containing labeled nucleotides, anddetecting incorporation of label into replicated DNA. Preferably, aradioactive nucleotide would be used to determine activation bymeasuring the extent of label incorporated into newly synthesized DNA.

The assay could be tailored to aid in the purification of factorspresent in CSF which help prepare nuclei for subsequent activation.Specifically, the assay would be performed without the addition of CSFextracts. Rather, various fractions of CSF extract would be obtained bystandard purification techniques, and used instead of CSF extract. Thosefractions which increase activation activity can then be furtherpurified.

Another use of a nuclear activation assay is to study male fertility bymeasuring the extent of activation of human sperm under differentconditions. Such studies can be used, for example, to examine techniquesto preserve sperm so the sperm can be later used in in vitrofertilization, to test sperm of infertile men to identify causes of maleinfertility (see, Brown et al., Yale Journal Of Biology And Medicine 65:29 (1992) (not admitted to be prior art), and test possible malecontraceptives.

In virtually all species of animals, sperm cells undergo two reactions,capacitation and the acrosome response, before reaching and fusing withthe egg surface. After the sperm nucleus enters an egg it undergoesseveral changes. The nucleus swells, acquires a nuclear envelope andlamina, replicates its DNA, and eventually fuses with the femalepronucleus. During this process, sperm basic proteins (histones andprotamines), are exchanged for embryonic histones.

It appears that in order for a sperm nucleus to respond to an eggcytoplasm it must first undergo some form of proteolytic digestion. Alikely site of necessary proteolytic digestion are non-histonecytoskeletal proteins. Possible contraceptives could target necessaryproteolytic enzymes. The affect of the contraceptive could be determinedby assaying the degree to which activation is inhibited. Possiblecontraceptives could also target other enzymes which may be needed foractivation.

Alternatively, the assay could be used to determine conditions whichresult in higher levels of activation thereby finding conditions whichenhance fertilization.

Specific uses of the nuclear activation assay include the following:

-   -   1) Assaying sperm cell treated under different conditions of        preparation, cryopreservation, capacitation and handling;    -   2) Assaying the affect of sperm cell enzymes (e.g., proteases,        nucleases, phosphatases, and kinases), including the inhibition        of sperm cell enzymes, on activation;    -   3) An assay to purify enzymes affecting activation;    -   4) Assaying the sperm from infertile individuals to determine if        infertility is due to problems with sperm nuclear activation;    -   5) Assaying the ability of specific drugs or reagents to enhance        or inhibit activation;    -   6) Assaying the affect of inhibitors or activators of sperm cell        enzymes on activation;    -   7) Assaying for the presence of a gene used to create a        transgenic animal; and    -   8) Assaying for the presence of viral genome, such as HIV        present in sperm.

The specific nuclear activation assay used to study fertility would betailored to study a particular aspect of activation. For example, toassay the effect of reagents on activation the sperm should be handledand prepared under mild conditions. As discussed above mild conditionsare useful in minimizing inadvertent activation of proteases ornucleases. To obtain sperm for the activation assay, fresh sperm samplesshould be first washed in isotonic saline solution under mildconditions. The sperm can then be stored by freezing in liquid nitrogenunder controlled conditions in the presence of a cryoprotectant. Martinet al., in PREIMPLANTATION GENETICS, Plenum Press, New York (Verinskyand Kuliev, eds, 1991).

Fresh or frozen/thawed sperm can be treated under conditions whichresult in capacitation as described by Martin et al. Supra. The spermmembrane is then permeabilized under mild conditions as described abovee.g., lysolecithin is used to permeabilize the membrane). The nuclei arethen recovered from lysed sperm by mild centrifugation in isosmoticbuffer. Nuclei are preferably pretreated as described above in SectionII (e.g., using a membrane permeabilizer, a protease, and a thiolreducing agent).

The nuclei can then be further pretreated (e.g., using CSF extractcontaining 100 μM Ca²⁺). The further pretreated nuclei are contactedwith activating egg extract and activation activity is measured usingstandard techniques such as using a labeled reagent (e., P³²-CTP) todetect DNA replication, and microscopic visualization of nuclearswelling. Additionally, in situ hybridization can be carried out todetermine the presence, number and location of particular DNA sequences.The effect of various reagents on nuclear activation can be determinedby adding reagents to the sperm before or after the various individualsteps of isolation, pretreatment, further pretreatment or contact withactivating egg extract.

VI. Retroviral Integration Assay

The assays described herein can be used to examine integration ofproviral nucleic acid, such as from HIV, into host DNA. The assays canbe carried out using a whole nucleus or a pseudonucleus. Such assays canbe used to identify target sites to inhibit proviral integration, and toderive anti-viral agents directed at such target sites.

A provirus is the double-stranded DNA form of a retrovirus. It issynthesized in the cytoplasm of a cell infected with a retrovirus byreverse transcription of the viral RNA. Integration of the provirus DNAinto the host cell genome is a critical step in the life cycle of allretroviruses, including HIV-1, and leads to viral expression and newvirus production. Thus, by blocking viral integration, viral propagation(e., viral multiplication and/or viral infection) can be inhibited.

An integration assay can be performed as follows:

1. A cell nucleus is pretreated to separate the nucleus from itssurrounding cytoskeleton to form a pretreated nucleus. The choice ofcell nucleus can be varied depending on the virus studied. Preferably,the cell nucleus will be obtained from a cell which is a natural hostfor the virus. Examples of cells susceptible to retroviral infectioninclude mammal and plant cells. Preferably, a human cell nucleus isused.

2. The pretreated nucleus is activated and incubated with a viralintegration complex. A viral integration complex contains the proviraldouble stranded DNA form of the viral RNA and material needed for viralintegration. Thus, the integration complex contains an integrase and maycontain other viral enzymes and proteins. An integration complex can beobtained by one skilled in the art using standard techniques. Forexample, a high speed supernatant of cells infected with a virus can beused as an integration complex. (Brown, et al., Cell 49: 347, 1987).Alternatively, an integration complex can be obtained from purifiedviral integrase and specific oligonucleotides having the viral sequencesneeded for integration (Engelman et al., Cell 67: 1211, 1991). Theintegration complex can be added to nuclei, chromatin or pseudonuclei,at different points in the cell cycle. For example, incubation can takeplace before (e.g., prior to activation), during the time that nucleiare swelling and forming nuclear envelopes in CSF extract and activatingextract, or during chromatin assembly, nuclear envelope formation orreplication of pseudonuclei.

3. Measuring integration of the viral nucleic acid into the host nucleicacid. The measurement can be carried out using standard techniques suchas through the use of hybridization probes targeted to viral nucleicacid sequences. The use of hybridization probes can be facilitated byamplification techniques such as PCR amplification. Preferably,unintegrated viral nucleic acid is separated from host nuclei acid priorto using the hybridization assay probe. A separation step is useful fordecreasing hybridization of probes to viral nucleic acid notincorporated into the host genome. Separation can be carried out, forexample, by centrifugation of nuclei through glycerol or electrophoresisof isolated nucleic acids.

Alternatively an integration assay can be carried out using apseudonucleus. A pseudonucleus can be constructed from a plasmid DNAtemplate which is then used as a target for retroviral integrationrather than intact activated nucleus. This approach has the advantagethat the oligonucleotide size of the plasmid genome is much smaller thanthat of a whole eukaryotic nucleus and the sequence of the plasmidgenome is either known or can be readily established.

A pseudonucleus can be constructed by adding plasmid DNA to a fresh orfrozen/thawed CSF extract (e.g., at 0.1-20 ng/μl). This material can beused immediately or frozen for latter use. The plasmid DNA can formchromatin in the CSF extract. (For example, see Sanchez et al., Journalof Cell Science 103: 907, 1992 and Wangh, Journal of Cell Science 93: 1,1989, describing such chromatin formation using plasmid FV1 dervivedfrom type 1 BPV in intact Xenopus eggs).

The assay is carried out by activating the chromatin and measuringintegration of viral nucleic acid. For example, the chromatin is dilutedinto an additional sample of CSF extract which is activated (e.g., bythe addition of 1.2 mM Ca²⁺). Activation triggers nuclear envelopeformation around the chromatin and causes the chromatin to replicate.

Thus, an activation assay can be performed using a pseudonucleus inplace of a pretreated nucleus as follows: 1) forming a pseudonucleus; 2)activating the pseudonucleus, and incubating with an integration complexcontaining viral nucleic acid before activation or at different timesafter activation; and 3) measuring integration of the viral nucleic acidinto the nucleic acid of the pseudonucleus.

Using an integration assay, it can be determined when viral integrationoccurs during the cell cycle, and if an agent is effective in inhibitingviral integration. For example, the importance of different stages ofthe cell cycle in viral integration can be evaluated using CSF oractivating extracts supplemented with DMAP, aphidicolin, and inhibitorsof type II topoisomerase. DMAP and aphidicolin block DNA replication butallow nuclear swelling and chromatin decondensation to proceed.Inhibitors of type II topoisomerase block chromatin decondensation,which requires type II topoisomerase activity. Examples of the use ofsuch drugs include the following:

1) If drugs such as DMAP and aphidicolin inhibit chromatindecondensation but fail to inhibit viral integration, then chromatindecondensation after mitosis is probably all that is necessary forintegration. In this instance, drugs could be designed to preventintegration during or prior to chromatin decondensation.

2) If integration (i.e., insertion of the proviral DNA into the targetgenome) and circularization (i.e., insertion of the proviral DNA intoitself) are both blocked by DMAP and aphidicolin, then on-going DNAsynthesis is probably required for viral integration. Accordingly viralintegration during DNA synthesis could be targeted. If DNA synthesis isrequired for proviral integration, it can then be determined whetherintegration occurs before or after the host genome target is replicated.For example, bromodeoxyuridine triphosphate (BrdUTP) can be added toreactions to increase the density of newly synthesized DNA strands.Samples can then be collected at the end of the S phase when genomereplication is complete. After electrophoretically removing allunintegrated viral molecules, the genomic DNA can then be cleaved with arestriction enzyme that recognizes two or more sites within the viralgenome. The preparation can then be fractionated, for example, by CsCldensity gradient centrifugation and probed for released segments of thevirus. If the provirus is inserted into the host genome beforereplication, the viral DNA will be recovered in the heavy/light densitypeak, along with virtually all the genomic DNA. On the other hand, ifthe provirus is inserted after its target sequence has replicated, theviral DNA will be in the light/light peak. After determining the timingof integration, drugs can be designed to inhibit integration and tested.

3) If integration takes place in the presence of DMAP or aphidicolin,but not both, this would indicate that DNA synthesis, per se, is notrequired for integration, but cell cycle-dependent properties of thecytoplasm influences integration.

Thus, using this application as a guide, one skilled in the art canidentify when, during the life cycle of a cell, viral integrationoccurs, target drugs to inhibit such viral integration, and assaywhether an agent inhibits viral integration. Agents which inhibitretroviral integration may be used as therapeutic agents to treat aperson infected with a retrovirus (such as HIV) or prevent an uninfectedperson from being infected with a retrovirus. A retroviral “therapeuticagent” refers to an agent which reduces, to some extent, the in vivopropagation of a retrovirus and preferably reduces, to some extent, oneor more of the symptoms associated with a retroviral infection.

VII. Microchamber Microscope Slide

Conversion and analysis of interphase nuclei to meiotic or mitoticchromosomes is facilitated using the microchamber microscope slide.Referring to FIG. 4, there is shown a microchamber microscope slide 10.The microchamber microscope slide allows very small amounts of expensiveand hard to come by reagents to be used sequentially on nuclei in situ.For instance, isolated nuclei can be placed into central microchamber 20which is formed tear-drop shaped, pretreated, swelled, converted tochromosomes, stained, and then read or analyzed without furthercentrifugation or complex manipulation.

A thin strip of PARAFILM^(•) wax 12, or other appropriate waterresistant plastic tape or like material, is annealed to a standardmicroscope slide 11 or coverslip 14. The microscope slide 11 isgenerally flat and rectangular-shaped with a top and bottom side. Thecoverslip 14 is generally flat and circular-shaped with a top and bottomside. The PARAFILM^(•) strip defines three wells connected by two narrowchannels. Center microchamber 20 is generally teardrop-shaped with agenerally rounded head end and a generally arrow shaped tail end. Thevolume of the microchamber is preferably between 5 μl and 50 μl, mostideally between 10 μl and 20 μl.

The head end of microchamber 20 is connected to a fill well 16 by anarrow entrance channel 18. The tail end of microchamber 20 is connectedto a drain well 24 by a narrow exit channel 22. The volume of theresulting wells is determined by the thickness of PARAFILM• strip 12 andthe size and shape of the wells; these parameters are adjustable.

Microchamber 20 is covered by a thin inverted coverslip 14. A thininverted coverslip is best suited for use with an upright compoundmicroscope. Other types of coverslips may be used. For example, anoptically thin coverslip is suited for use with an inverted compoundmicroscope.

In the preferred mode of operation, coverslip 14 completely coversmicrochamber 20 leaving fill well 16 and drain well 24 substantiallyuncovered. A sample of cells, nuclei, or other material, is pipetted tothe wide part of the microchamber. The microchamber is then covered witha coverslip which is caused to adhere to the upper surface of thePARAFILM•strip 12 by applying two small drops of paraffin oil. Theoverlaying coverslip can be siliconized to minimize sticking of waterand other materials to this surface.

Microchamber 20 is filled by capillary action by placing fluid in fillwell 16. Excess fluid is then removed from both the fill well and drainwell 24. The microchamber is flushed by placing fluid in the fill welland then sucking the fluid through the microchamber by capillary actionachieved by touching blotting paper to the edge of the drain well.

The general teardrop shape enhances flushing of microchamber 20. For a10 μl microchamber as little as 20 μl of fluid is sufficient to clearthe microchamber. If necessary, the coverslip 14 can be removed and thefill channel 18 and exit channel 22 sealed with a small bead of siliconestopcock grease. Material can then be recovered from the microchamber.

The microchamber microscope slide is extremely versatile. It can besterilized, placed in tissue culture medium, and used as a growingsurface for cells. Further, it is possible to increase the depth of thetwo side wells while leaving the microchamber shallow. Each of the sidewells could be covered with their own lid. One of the side wells couldbe filled several millimeters deep with tissue culture medium while theother well is left unfilled. Tissue culture medium would then flowthrough the microchamber across the cells until the two side wells reachequilibrium, the exact flow rate being adjustable. Further potentialuses include the following: (1) analysis of growing cells; (2) analysisof isolated cells, particularly fetal blood cells; (3) analysis of cellnuclei, or other subcellular particles, organelles or materials; and (4)analysis of material of non-living origin.

The microchamber microscope slide allows analysis of material byessentially all light microscopy staining techniques including thefollowing: (1) fluorescent microscopy of incorporated precursors,antibody staining, nucleic acid hybridization techniques; (2)conventional histological staining procedures; and (3) staining based onenzymatic amplification of molecular signals.

The microchamber microscope slide also allows analysis of biologicalmaterial by incorporation of radioactive precursors, followed byautoradiographic detection of the incorporated precursors.

The microchamber microscope slide also allows “on-line” microscopicobservation of material being treated or altered by fluids flowingthrough the microchamber. In particular, the microchamber microscopeslide is ideally used to both isolate and analyze fetal cell frommaternal blood. Isolation may be achieved by first coating themicrochamber with the appropriate antibody to fetal cells, or theiralready isolated nuclei, and the microchamber is otherwise not sticky.The microchamber itself selects and holds the fetal cells, or nuclei,while the maternal cells, or nuclei, are washed away. The fetal cells,or nuclei, can then be fluorescently tagged in situ and their positionsidentified even before starting in vitro nuclear swelling and chromosomeformation. The fetal cells or nuclei, can then be activated using theappropriate treatments described herein.

VIII. Activation Kits

The technology disclosed in the present invention can be used to produceactivation kits useful for clinical activation of nuclei and scientificresearch. These kits are particularly useful for prenatal screening.Uses of an activation kit to aid in scientific research includefacilitating the study of complex biochemical activities including theassembly of nucleosomes and chromatin on plasmid or viral DNA, formationof eukaryotic nuclear envelopes surrounding nuclear templates,semi-conservative replication of double stranded DNA within eukaryoticnuclei, conservative repair replication of single stranded DNAindependent of nuclear envelope assembly, activation of quiescent cellnuclei, nuclear envelope breakdown, condensation of chromatin intochromosomes, formation of meiotic and mitotic spindles, regulatedtranscription of eukaryotic genes, and protein synthesis.

A basic activation kit comprises frozen activating egg extract andfrozen CSF extract. These extracts are prepared based upon the methodsdescribed in the present invention. Preferably the kit contains frozenactivating egg prepared from eggs having an elevated DNA synthesisactivation activity. More advanced kits contain various supplementswhich aid in activation. The various supplements are either in separatecontainers present in the frozen activating egg extract or frozen CSFextract.

Preferably, these supplements are in separate containers. Usefulsupplements includes CaCl₂, nocodazole, β-glycerol-PO₄,phosphodiesterase inhibitor (e.g., caffeine), cAMP, protein kinaseinhibitor (e.g., DMAP). Preferably, the activation kit contains amicrochamber microscope slide.

The activation kits could also be supplemented with reagents used tostudy activation in general or determine the extent of genomereplication. Useful supplements for these activities include radioactivenucleotides, biotinylated nucleotides and different dyes (e.g.,biotin-Texas Red streptavidin and Hoechst).

IX. Cloning Whole Animals from Somatic Cell Nuclei

The procedures disclosed by the present invention, to activate nuclei,are also useful for preparing a nucleus for subsequent transplantationinto an egg for the purpose of directing the development of a neworganism. Prior to nuclear transplantation, the nucleus to betransplanted is activated in vitro. The activated nucleus is thentransplanted into an egg whose own nucleus has either been removed orfunctionally inactivated. The egg subsequently develops into an neworganism under the direction of genetic information contained in thetransplanted nucleus. Uses of cloning somatic cell nuclei include,creation of a clone of genetically identical animals, cloning animalshaving favorable attributes, and producing more animals which are indanger of becoming extinct.

A difficulty in cloning somatic cell nuclei from mammalian species isthat these nuclei are imprinted with patterns of gene structure andfunction (e.g., DNA methylation patterns) which differ from sperm andegg nuclei patterns. Thus, it is necessary to reprogram somatic cellnuclei before cloning to eliminate the different patterns. Prioractivation of somatic cell nuclei in an appropriate egg extract beforetransplanting should allow for the necessary reprogramming to enable atransplanted nucleus to give rise to either a complete, or substantiallycomplete new organism.

Cloning using a somatic cell nucleus comprises three steps; (1)activating the somatic cell nucleus, (2) preparing a recipient egg, and(3) transplanting the somatic cell nucleus into the egg. The first stepis preferably carried out using the improved procedures, disclosedabove, to activate a nucleus. Preferably isolation, pretreatment,further pretreatment, and contact with activating egg extract arepreformed under conditions where the activated nucleus has a high DNAsynthesis activation activity.

Preparation of a recipient egg will vary depending upon the egg source.The egg source should be treated in a manner to prevent activationbefore nuclear transplantation. Procedures to prepare mammalian eggs,such as those described by Martin et al. supra, are know in the art.

Preparation of a recipient egg includes destroying the egg's pronucleus.Destruction or removal of the egg's own nucleus guarantees that the eggsgenetic material (DNA) does not contribute to the growth and developmentof the newly cloned individual. One method of destroying the pronucleusis by using ultraviolet light as described by Gurdon, in METHODS IN CELLBIOLOGY, XENOPUS LAEVIS:PRACTICAL USES IN CELL AND MOLECULAR BIOLOGY,36: 299-309, Academic Press, California. (Kay and Peng eds., 1991).Alternatively, the egg pronucleus can be surgically removed byprocedures known in the art such as those described by King, in METHODSIN CELL PHYSIOLOGY 2: 1-36, Academic Press, New York (D. M. Prescott,ed., 1966), and McGrath and Solter, Science 220: 1300-1319 (1983)

Nuclear transplantation can be carried out by standard techniques. Thesetechniques, vary depending upon the species, and are known in the art.

It should be possible to clone Xenopus in the following manner: nucleifrom Xenopus red blood cells are isolated, pretreated, and furtherpretreated. Nuclei are then activated by contact with an activating eggextract. The nuclei are then activated to different stages in the cellcycle (e.g., S-phase, G2, etc.), and transferred to recipient preparedXenopus eggs.

Recipient Xenopus eggs are prepared for nuclear transplantation byhardening using Ca²⁺ (as described above), and then irradiating withultraviolet light to destroy the egg's genome. One to two activatedsomatic nuclei, in 20 to 50 nanoliters are then microinjected into theXenopus egg, into the clear cytoplasmic region that lies approximately400 microns below the animal pole of the egg. The egg is then incubatedunder conditions that permit cytoplasm rotation. These conditions can beconveniently obtained by floating the egg on Metrizamide^(•). Rotationof the egg cytoplasm relative to the egg cortex is important forestablishment of the proper dorsal/ventral axis of the developingvertebrate embryo.

X. EXAMPLES Example 1 Further Induction Optimization

This example describes additional experiments carried out to furtherdetermine the optimal induction time for an activating egg extract.Protein synthesis during the early part of the first cell cycle inactivated eggs or egg extracts is required for preparation of anactivating extract capable of efficient and complete genome replication.The required proteins can either be synthesized in intact eggs beforepreparation of extracts or in extracts including frozen/thawed extracts.As noted above, activating egg extract should be prepared from extractsinduced for more than 10 minutes to enhance DNA synthesis activationactivity.

It was found that proteins synthesized during the first 28-30 minutes inintact eggs (incubated at 20° C.) or during the first 60-80 minutes in afreshly prepared and activated extract (incubated at 25° C.), promotesubsequent DNA replication. In contrast, proteins synthesized later inthe first cell cycle, i.e., after replication is underway, inhibit DNAsynthesis. The changes in DNA synthesis can be detected as alterationsin the time which DNA synthesis starts, the initial rate of replication,and the overall amount of replication.

The amount of CaCl₂ used to induce a freshly prepared CSF extractregulates whether or not the extract exits meiotic metaphase arrest,traverses the first interphase, and re-enters the first M-phase. Asjudged by measurements of histone H1 kinase activity, fresh CSF extractinduced by the addition of 3 mM CaCl₂ exits meiotic metaphase, entersinterphase, but fails to enter mitosis-I. In contrast, CSF extractinduced with 1.2 mM CaCl₂ exits meiotic metaphase, enters interphase,and then proceeds into mitosis-I, as indicated by a second peak in H1kinase activity.

For the purpose of comparison, extracts were prepared from eggs inducedand incubated at 20° C. for varying lengths of time before beingcrushed. In all cases the eggs were amassed, induced, washed, crushed,and extracts were prepared as described for prepared activating extractswith the following modifications: (1) all tubes and pipette tips used toprepare egg extracts were first treated with 1% diethylpyrocarbonate todestroy ribonuclease activity and (2) all the steps in extractpreparation were carried out using plastic gloves to avoid ribonucleasecontamination. Extracts were frozen on an aluminum block, chilled withliquid nitrogen. and then thawed at a later time prior to being used.DNA synthesis was followed by incorporation of P³²dCTP, followed byelectrophoresis and phosphoimager analysis.

The results demonstrate that optimal DNA synthesis activating extractsare obtained by synchronously inducing batches of eggs and incubatingthem for 28-30 minutes at 20° C. Of the time periods tested the 28-30minute extracts initiated nuclear replication earliest, synthesized DNAfastest, and replicated more DNA, then egg extracts induced for 10minutes, 22 minutes, 34 minutes or 40 minutes. The overall order forearlier nuclear replication, faster DNA synthesis, and extent of DNAreplication was as follows: 10 minutes<22 minutes<25 minutes<28minutes>34 minutes>40 minutes. Because the cell cycle of the egg is sorapid, even small differences in the length of incubation period or thetemperature of incubation result in suboptimal extracts.

It was also determined that maximal replication, even in thefrozen/thawed 28-30 minute extract, depends on continuing proteinsynthesis during the first 30 minutes of the in vitro incubation.Activating egg extract were prepared as described above, induced for 28minutes. Cycloheximide was added just prior to induction, or 30 minutesafter induction. Maximal DNA replication was observed for control (nocycloheximide) and cycloheximide added 30 minutes after induction, whilezero minute cycloheximide addition resulted in significantly less DNAreplication. This suggests that the proteins required for efficientreplication are relatively unstable but are abundantly synthesized frommRNAs recruited onto polysomes during the first 28-30 minutes followingegg induction.

Example 2 cAMP Supplemented Activating Egg Extract

The affect of cAMP on DNA replication in activated Xenopus red bloodcells was determined. Xenopus nuclei were isolated and pretreated by amethod based on Coppock et al., Developmental Biology 131: 102 (1989),as follows: Xenopus blood was obtained from females by cardiac punctureand collected using a syringe half-filled with Barth's solution (88 mMNaCl, 2.3 mM KCl, 0.82 mM MgCl₂ and 10 mM Hepes, pH 7.4) containingheparin (10 mg/ml); the blood was immediately diluted into 10 ml ofice-cold 0.6×SSC (1×SSC is 0.15 M NaCl, 0.015 M Sodium citrate, pH 7.0)containing 0.1 mg/ml heparin, 0.1 mM TPCK (N-tosyl-L-phenylalaninechloromethyl ketone), 0.1 mM TLCK (Na-p-Tosyl-L-lysine chloromethylketone), 0.05 mM PMSF (phenylmethylsulfonyl fluoride), 5 μg/mlleupeptin, and 31.25 mM Na₂S₂O₅; bleeds containing clots, even smallones, were rejected; diluted blood was underlaid with 0.5 volumes of icecold Metrizamide^(•) (refractive index of 1.3660 in 0.6×SSC) andcentrifuged at 180 g for 10 minutes at 4° C., red blood cells pelletedbelow Metrizamide^(•) while white cells banded above Metrizamide^(•);the red cell pellet was resuspended using 0.6×SSC and centrifuged inMetrizamide^(•) four more times to obtain erythrocytes of greater than99.9% purity; cells were washed three times in NIB and resuspended at2×10⁸ cells/ml; cells were then resuspended in NIB:glycerol (7:3) andfrozen in aliquots of 100 μl in liquid nitrogen; before using, frozencells were thawed, diluted to 4×10⁷ cells/ml in NIB at 23° C., and addedto an equal volume of NIB containing 80 μg/ml lysolecithin (40 μg/mlfinal concentration) and 0.6 μg/ml trypsin (0.3 μg/ml finalconcentration) (the trypsin used in this example, and the other examplesdescribed herein, was Sigma brand Type XIII trypsin, TPCK treated fromBovine Pancrease, approximately 11,000 units/mg solid); after 5 minuteslysolecithin and protease treatment was stopped by adding soybeantrypsin inhibitor to a concentration of 30 μg/ml and bovine serumalbumin to a final concentration of 0.4%; the resulting nuclei werecentrifuged at 800 g at 0° C. for 10 minutes, washed twice in NIB,resuspended with ice-cold NIB and kept on ice.

Isolated and pretreated nuclei were added at 200 nuclei/μl to 550 μlthawed “prepared activating egg extract” supplemented with 5 μg/mlnocodazole, 250 μg/ml cycloheximide, 10 μCi P³²-dCTP, 10 mM creatinephosphate, and 10 μg/ml creatine phosphokinase. Cyclic-AMP was thenadded to separate aliquots to yield final concentrations of 0.0 μM, 0.1μM, 1.0 μM, or 10 μM.

Each aliquot was warmed to 23° C. and sampled over time to determineP³²-dCTP incorporation into replicated DNA. At each time point, a 7 μlaliquot was taken, frozen on dry ice, and later thawed and digested bythe addition of 10 μl replication sample buffer (80 mM Tris (pH 8.0), 8mM EGTA, 0.13% phosphoric acid, 10% Ficoll, 5% SDS, 0.2% bromphenolblue) containing proteinase K (1.0 mg/ml) for 2 hours at roomtemperature. Incorporated radioactivity was analyzed by electrophoresison a 0.8% agarose gel (50V, 20 hours) followed by vacuum drying the geland counting on a Betascope.

As indicated by Table 2, the addition of 10 μM cAMP inhibits DNAreplication in activated nuclei as compared to DNA replication occurringwithout any cAMP. DNA replication increased with 0.1 μM and 1.0 μM cAMP.A greater increase was seen with 1.0 μM than with 0.1 μM cAMP. Thus,cAMP can be used to increase the activation activity of activating eggextracts. A concentration of approximately 0.3 μM, was used insubsequent studies. TABLE 2 Cpm Incorporated Micromoles of Cyclic AMPAdded After X Minutes 0.0 0.1 1.0 10 0 14 14 10 43 45 37 51 14 34 90 7368 56 31 135 288 290 564 35 180 839 1,141 2,316 85 240 1,556 2,945 4,484168 300 2,954 2,692 5,504 571

Example 3 Caffeine Supplemented Activating Egg Extract

The effect of caffeine on DNA replication in activated Xenopus red bloodcells was determined. The experimental conditions used were as describedin Example 1 with the following changes: the concentration of cAMP wasset at 0.3 μM and caffeine was added to the activating egg extract to aconcentration of either 0.2 mM, 1.0 mM, or 5.0 mM.

As illustrated by FIG. 1, caffeine at 1.0 mM in the presence of 0.3 μMcAMP gave the highest initial rate and extent of DNA replication inactivated nuclei. Thus, caffeine can increase the activation activity ofan activating egg extract.

Example 4 CSF Extract Supplemented with DMAP and Treated with ActivatingExtract

Addition of 6-dimethylaminopurine (DMAP) to CSF extracts was used tofurther pretreat Xenopus erythrocyte nuclei and stimulate subsequent DNAreplication in activating egg extract. DMAP can be used to inhibitnucleic acid synthesis and protein kinase activity. Xenopus erythrocytenuclei were isolated and pretreated as described in Example 1 above, andincubated in thawed “prepared CSF extract” supplemented with 80 mMβ-glycerol-PO₄, and 5 μg/ml nocodazole at a concentration of 2000nuclei/μl. Further pretreatment was carried out by incubation for 30minutes at 4° C., then 30 minutes at 25° C., hen 60 minutes at 4° C.Half the samples were supplemented with 5 mM DMAP before addition of thenuclei. After the two hours of incubation, each sample was diluted with9 volumes of activating egg extract, supplemented with 5 μg/mlnocodazole (this dose of nocodazole slows down the rate of replication)and approximately 160 μCi/ml P³²-dCTP. Aliquots were removed over timeto measure DNA replication.

As illustrated by FIG. 2, the addition of DMAP to CSF extracts enhancedthe ability of the CSF extract to stimulate subsequent DNA replicationin activating egg extract. DMAP decreased the lag time before the onsetof replication and increased the initial rate and total amount of DNAsynthesis.

Example 5 Warm-Then-Cold Regime

Various warm-then-cold regimes used as part of a further pretreatmentincreased DNA replication in activated nuclei. Thawed Xenopuserythrocyte nuclei (isolated and pretreated as in Example 1 above) wereadded at 2000 nuclei/μl to thawed “prepared CSF extract,” supplementedwith 80 mM β-glycerol-PO₄. The mixture was incubated using variouswarm-then-cold regimes. At the end of each incubation period samples wasdiluted with 9 volumes of “prepared activating egg extract” supplementedwith 5 μg/ml nocodazole and P³²-dCTP. Samples were removed over time tomeasure the extent of DNA replication.

As shown by the data represented in FIG. 3, the following warm-then-coldregimes stimulated subsequent DNA replication: 30 minutes at 25° C., and90 minutes at 4° C.; 30 minutes at 4° C., 30 minutes at 25° C., and 60minutes at 4° C.; 60 minutes at 4° C., 30 minutes at 25° C., and 30minutes at 4° C. Incubation for 90 minutes at 4° C., and 30 minutes at25° C. was not as effective as incubation regimes that had a warm periodfollowed by a cold period. Thus, nuclei activation is preferablyperformed using a warm-then-cold regime.

Example 6 Activation Using Frozen/Thawed Extracts

Activation of Xenopus red blood cell nuclei was studied usingfrozen/thawed CSF extracts and frozen/thawed activating egg extract.Xenopus red blood cell nuclei were isolated and pretreated as describedin example 1. These nuclei were further pretreated at 2000 nuclei/μl inthawed “prepared CSF extract” supplemented with 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, 5 μg/ml nocodazole, 80 mMβ-glycerol-PO₄, 100 μM CaCl₂ and incubated using a warm-then-cold formatof 60 minutes at 25° C. followed by 60 minutes at 4° C.

After further pretreatment, samples were diluted with 9 volumes ofthawed “prepared activating egg extract” containing 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, and incubated with either:A) 200 μCi/ml P³²-dCTP; B) 16 μM biotin-11-dUTP, 16 μM MgCl₂; or C) noadditions.

At various time intervals an aliquot of each incubation was treated asfollows:

A) P³²-labelled samples were treated with sodium dodecyl sulfate (SDS),proteinase-K, and then analyzed on agarose gels to determine DNAreplication. Total incorporated radioactivity was measured using aMolecular Dynamics phosphoimager. The sizes of the radioactive moleculeswere observed and photographed on X-ray film.

B) Biotin labelled nucleic acid was used to visualize replicated nuclearDNA. Biotin labelled samples were fixed by mixing into the samplesapproximately 40 volumes of freshly prepared 1.0 mM ethylene glycolbis-(succinic acid N-hydroxysuccinimide ester) (EGS) and incubating at37° C. for 30 minutes. Fixed nuclei were stored at 8° C. for 48 hoursand then centrifuged onto glass coverslips (2000 rpm, at 4° C. for 15minutes) through a 25% glycerol layer. The glycerol layer was removedand the samples were stained with Texas Red-Streptavidin (Gibco BRL,diluted 1:40 in PBS). Coverslips were then washed with buffered salineand stained with 1.0 μg/ml Hoechst 33258 stain (for total DNA). Eachsample was examined and photographed at 60× using an Olympus opticalsystem. Using these conditions: 1) nuclear envelopes were detected underphase optics as a dark line around the nucleus; 2) total nuclear DNA wasobserved under fluorescent optics as Hoechst positive (blue) staining;and 3) newly replicated biotinylated DNA were detected as Texas Redpositive (red) staining.

C) Samples from incubate (C) were used to measure histone H1 kinaseactivity during the course of the experiment.

As judged by both P³²-dCTP incorporation and biotinylated-dUTPincorporation, new DNA replication in erythrocyte nuclei was highlysynchronous and efficient. Replication began at 30-40 minutes ofincubation and was completed by 80-90 minutes of incubation. Noadditional DNA synthesis was observed between 90-140 minutes. After 140minutes DNA replication resumed. The initial rate of DNA synthesis inthis system using a frozen/thawed activating egg extract is onlyslightly slower than that using fresh activating egg extract.Furthermore, it appears that replication of the entire genome wasachieved.

As judged by nuclear morphology and staining, swelling was observedabout 20 minutes after addition of activating egg extract (T=20). AtT=20 no DNA replication was observed. DNA synthesis andbiotinylated-dUTP incorporation were both first observed at T=40.Nuclear swelling continued until T=80 at which time nuclear condensationand nuclear envelope breakdown began. Photographs of 10 or more nucleiat each time point revealed that virtually all nuclei in each samplewere activated at the same time and in the same manner. Hoechst stainingand biotin labelling revealed that nuclear DNA was first highlycompacted (T=0), became more diffuse during the period of swelling andreplication (T=20 to T=80), and then condensed into chromosome likestructures (T=100 to T=180). Nuclear envelope breakdown occurred atT=100 to T=120 minutes, but mitotic spindle formation was not observedin these samples. This was likely due to the presence of low levels ofnocodazole (0.5 μg/ml). At T=160 many of the nuclei appeared under phasecontrast to have nuclear envelopes suggesting they entered a secondinterphase. All nuclei at T=180 had distinct chromosome-like structuresindicating that they entered a second mitosis. DNA synthesis (P³²-dCTPincorporation) resumed between T=140 and T=160 and then stopped at T=160in accord with a second S-phase followed by a second mitosis.

Histone H1 kinase levels were low at the start of S-phase (T=40) androse gradually thereafter. Correlations of DNA synthesis, nuclearmorphology, and H1 kinase levels suggested that a threshold level of H1kinase leading to nuclear envelope breakdown and first mitosis wasreached at T=100. H1 kinase levels continued to rise until T-180,despite the fact that DNA synthesis resumed at T=140-160.

First mitosis occurred relatively early (T=100 to T=120) and was notaccompanied by DNA fragmentation. These observations are consistent withthe view that genome replication was complete in this experiment.Agarose gel analysis of the p³²-labelled DNA demonstrated that virtuallyall newly synthesized DNA was initially of very high molecular weight(HMW), some of this material was then converted to pieces of a ratheruniform moderate molecular weight (MMW). DNA pieces of the MMW size arenot degradation products and reflect a fundamental unit of DNA packagingin condensing chromosomes. MMW may be due to experimental interruptionof topoisomerase II dependent deconcatenation of replicated DNA loops.

In summary this experiment demonstrates, an in vitro system usingfrozen/thawed CSF extracts, and frozen/thawed activating egg extractsprepared from Xenopus eggs. In this system nuclei swell, acquire newenvelopes, and cycle through at least one complete S phase followed byone complete M phase. Only a limited amount of DNA synthesis takes placein a second S-phase. This system permits highly synchronous activationand cycling of quiescent cell nuclei, and is directly applicable to theactivation of non-dividing human cells such as fetal cells includingkeratinocytes, trophoblasts, erythrocytes and leukocytes, and spermnuclei.

Example 7 Comparison of High Speed Versus Low Speed CSF Extract

This example compares nuclei activation using a pretreatment in eitherlow speed or high speed “prepared CSF extract.” Activation was carriedout using frozen/thawed activation extract induced for 28 minutes.Nuclei activation was assayed by measuring DNA replication and HistoneH1 kinase activity.

Xenopus erythrocyte nuclei were prepared as described in Example 2 andincubated in either a low speed prepared “CSF extract” or a high speed“prepared CSF extract.” In both cases nuclei were then diluted into 9volumes of a 28 minute induced activating egg extract. For each incubateone set of samples were collected to measure histone H1 kinase activity,another set of samples containing P³²-dCTP was used to measure DNAsynthesis. The nuclei incubated in the high speed CSF extract replicatedand progressed through the cell cycle more synchronously than thoseincubated in low speed CSF extract.

Example 8 Use of Diluted CSF Extract Supplemented With DMAP

This example illustrates the use of diluted CSF extract supplementedwith DMAP to achieve nuclear envelope formation and nuclear structure inthe absence of DNA synthesis. Xenopus erythrocyte nuclei were isolatedand pretreated using lysolecithin and trypsin as described in Example 2above. “Prepared CSF extract” was made using high speed centrifugationand frozen by spotting the extract, made 7.5-10% (v/v) glycerol, as a 20μl droplet onto a block of aluminum immersed in liquid nitrogen.Aliquots of the extract were thawed on ice and supplemented with 10 mMcreatine phosphate, 10 μg/ml creatine phosphokinase, 80 mMβ-glycerol-PO₄, and 0.1 mM CaCl₂. While still on ice the CSF extractreceived a small volume ({fraction (1/33)}^(rd)) of DMAP to a finalconcentration of 5 mM, and was then diluted with different amounts of EBbuffer as follows: mixture 1, 100%=extract only no EB; mixture 2, 75%=3volumes extract+1 volume EB; mixture 3, 50%=1 volume extract+1 volumeEB; and mixture 4, 25%=1 volume extract+3 volumes EB.

Each of these mixtures was warmed to 25° C. and incubated for 15minutes. Pretreated nuclei were then added in {fraction (1/10)} thevolume to a final concentration of 2000 nuclei/μl. Samples from eachincubate were taken immediately (0), 60, 90, 120 minutes later andfixed, examined, and photographed. Nuclei treatment with dilutedCSF-DMAP resulted in greater swelling than nuclei treated with undilutedCSF-DMAP. Nuclei treated with mixture 3 had a larger extent of nuclearswelling, nuclear envelope formation, and chromatin decondensation, thannuclei treated with the other mixtures. Additional experiments usingP³²-dCTP and biotinylated-dUTP demonstrated that no DNA synthesis tookplace during the process of nuclear swelling described above.

Example 9 Use of Diluted CSF Extract Supplemented With DMAP, MgCl₂ andEGTA

This example illustrates the effect of diluted CSF extract supplementedwith DMAP, MgCl₂ and EGTA on nuclear envelope formation, swelling, andchromatin structure. Nuclei were treated as described in Example 7 priorto the addition of CSF extract. The CSF extract (prepared as in Example7), while still on ice was supplemented as follows: 5 mM DMAP, 16 μMBiotinylated-dUTP and 16 μM MgCl₂. The supplemented extract was dilutedwith an equal volume of EB buffer containing 5 mM potassium EGTA, pH 7.This mixture was warmed to 25° C. and incubated for 15 minutes.Pretreated nuclei were then added in {fraction (1/10)} the volume to afinal concentration of 2000 nuclei/μl. Samples from each incubate weretaken immediately 0, 15, 45, 60, and 90 minutes later and were fixed,examined, and photographed.

The 50% CSF-DMAP, supplemented with EGTA and MgCl₂, caused pretreatederythrocyte nuclei to rapidly swell and acquire a nuclear envelope. Nobiotin incorporation into DNA was observed. Thus in contrast to Example7, swelling took place in the absence of DNA synthesis. In addition, theDNA observed in this example was more compacted than the DNA observed inexample 8. The difference between this example and example 8 is likelydue to the alteration of CSF extract cation concentration, andcomposition. For example, the EGTA may chelate the Ca²⁺ thereby loweringthe Ca²⁺ while additional Mg²⁺ is added to increase the Mg²⁺concentration.

Example 10 Microchamber Microscope Slide

This example illustrates the use of the microchamber microscope slide toanalyze and activate nuclei. Xenopus erythrocyte nuclei were isolatedand pretreated as described above in Example 1. These nuclei, in NIBbuffer, were allowed to settle onto the lower surface of severalmicrochamber microscope slides. A coverslip was placed over each sampleand sealed using oil along the sides. Nuclei were further pretreatedusing thawed “prepared CSF extract” made 80 mM in β-glycerol-PO₄ andsupplemented with 10 mM creatine phosphate and 10 μg/ml creatinephosphokinase. Ten microliters of CSF extract was allowed to flow intoeach well and the microchamber microscope slide was then subjected tothe following warm-then-cold treatment; 30 minutes on ice, 30 minutes at25° C., and 30 minutes on ice. After the warm-then-cold treatment, theCSF extract in each well was displaced by the addition of 20 μl freshly“prepared activating egg extract” containing biotinylated-dUTP. Themicrochamber microscope slides were then warmed to 25° C. At varyinglengths of time the incubations were stopped by rinsing the microchamberwith 75 μl of an appropriate buffer containing Texas red streptavidin(for detection of incorporated biotin), followed by staining withHoechst stain for detection of total DNA. The nuclei were photographedat a magnification of 60× using fluorescent optics.

Red blood cell nuclei before pretreatment were small and compact. Themajority of nuclei were separated from one another indicating they werenot damaged or clumped during isolation. Red blood cell nuclei at theend of further pretreatment in CSF extract were attached to the surfaceof the microchamber microscope slide and remained small and highlycompact.

Red blood cell nuclei 30 minutes after addition of activating eggextract swelled dramatically, and were attached to the surface of theslide. Texas red streptavidin staining of these nuclei demonstrated thelack of DNA replication.

Nuclei after 85 minutes of incubation in activating egg extract wereswollen. As seen by Texas red streptavidin staining, these nuclei weresurrounded with a nuclear envelope and initiated DNA replication.

After 150 minutes of incubation in fresh activating egg extract DNAreplication was complete and the nuclei entered mitosis. As a result ofentering mitosis, the nuclear envelopes dissembled and the DNA condensedinto chromosome-like structures which remained attached to the surfaceof the microchamber microscope slide.

These results demonstrate the utility of a microchamber microscope slidein nuclei activation. Using the methods and products disclosed in thepresent invention nuclei were conveniently activated on a microchambermicroscope slide.

Example 11 Activation of Nuclei from Fetal Red Blood Cells Isolated fromthe Umbilical Cord

The activation of human fetal red blood cells using activating eggextract is described below. Human fetal red blood cells were preparedfrom umbilical cord blood, pretreated with lysolecithin and trypsin, andcontacted with activating egg extract.

Human fetal red blood cells were isolated from umbilical cord blood andfractionated into a nucleated cell fraction and a non-nucleated cellfraction as described by Bianchi et al., Proc. Natl. Acad. Sci. USA 87:3279 (1990). A sample of neonatal umbilical cord blood was drawn into avacuum tube containing anticoagulants, the blood was diluted 1:1 withHank's balanced salt solution (HBSS) (Hanks and Wallace, Proc. Exp.Biol. Med. 71: 196 (1949)), layered over a Ficoll/Hypaque column(Pharmacia) and spun at 1400 rpm for 40 min at room temperature. Themononuclear cell layer was recovered and washed twice by centrifugationin HBSS. The cells were then washed several times in NIB buffer (250 mMsucrose, 25 mM NaCl, 10 mM Pipes, 1.5 mM MgCl₂, 0.5 mM spermidine, and0.15 mM spermine, pH 7.0); the resulting cell pellet was suspended inNIB:Glycerol (7:3) and frozen in liquid nitrogen as 100 μl aliquotscontaining 6.3×10⁶ cells.

Frozen cells were thawed at room temperature and put on ice, washedtwice with NIB, diluted to 4×10⁷ cells/ml in NIB at 23° C., and added toan equal volume of NIB containing 80 μg/ml lysolecithin and 0.6 μg/mltrypsin; lysolecithin and trypsin treatment was halted after 5 minutesby adding soybean trypsin inhibitor to a concentration of 30 μg/ml andbovine serum albumin to a final concentration of 0.4%. Isolated nucleiwere added directly to thawed “prepared activating egg extract” to aconcentration of 200 nuclei/μl, supplemented with 10 mM creatinephosphate, 10 μg/ml creatine phosphokinase, 5 μg/ml nocodazole, 0.3 mMcAMP, and 1 mM caffeine. One aliquot of this sample was supplementedwith P³²-dCTP at approximately 200 μCi/ml and used to measure DNAreplication. A second aliquot was sampled periodically for fluorescentmicroscopic examination of nuclei after fixation and staining withHoechst dye (as described above).

Human red blood cell nuclei treated in the manner described aboveswelled significantly during the first 90 minutes and initiated DNAsynthesis. DNA synthesis continued for approximately 4.0 hours afterwhich nuclear chromatin condensed. However, the observed kinetics of DNAsynthesis indicated that complete genome replication was not achieved inthis experiment. The failure to achieve complete genome replication wasprobably due to the failure to further pretreat the isolated humannuclei in CSF extract and because activating egg extract contained arelatively high level of nocodazole, i.e., 5 μg/ml.

Despite the difficulties encountered, the formation of metaphasechromosomes demonstrates that the present invention can be used toactivate non-dividing human nuclei. The non-dividing human nucleiactivated analogously to Xenopus erythrocyte nuclei. Therefore, thevarious improvements described in the present invention, such as furtherpretreatment in CSF extract, a warm-then-cold regime and the addition of100 μM CaCl₂, which result in increasing the activation of non-dividingXenopus erythrocyte are applicable to activate non-dividing humannuclei.

Example 12 Activation of Nuclei of Fetal Red Blood Cells Isolated FromFetal Liver

This example illustrates the use of the products and methods describedherein to determine preferred activation conditions and activate bloodcells isolated from fetal liver. Mononucleated cells isolated from fetalliver, studied in this example, were predominately fetal blood cells asjudged by their red color. The following steps were preformed:

Step 1: Isolation of mononucleated human liver cells. Mononucleatedhuman cells were isolated from human fetal liver by gently trimming thetissue and then homogenizing it between two glass slides. The cells werecollected by suspension in phosphate buffered saline and thentransferred to a centrifuge tube. 2 ml of Ficoll was layered under thecell suspension which was then centrifuged at 2000 rpm for 20 minutes.The red mononuclear cells (upper layer) containing predominantlyerythroid blood cells were collected, diluted with phosphate bufferedsaline and centrifuged gently to pellet the cells. Cell pellets wereresuspended and pelleted one more time in phosphate buffered saline andthen resuspended in RPM1 tissue culture medium.

The cells were frozen in liquid nitrogen. To prepare cells for freezingin liquid nitrogen, the cells were pelleted by gentle centrifugation,resuspended, and centrifuged again in Hank's balanced salt buffercontaining protease inhibitors (TPCK 0.1 mM, TLCK 0.1 mM, PMSF 0.05 mM,and leupeptin 5 μg/ml) at 4° C. The resulting supernatant was clearindicating the absence of hemolysis. The pellet was resuspended in 1-1.5ml of NIB containing protease inhibitors (TPCK 0.1 mM, TLCK 0.1 mM, PMSF0.05 mM, and leupeptin 5 μg/ml), the volume was brought to 10 ml. Thesuspension was then spun at 1000 rpm for 10 minute at 4° C., again nohemolysis was observed, and resuspended to final volume of 5 ml in NIBcontaining protease inhibitors (TPCK 0.1 mM, TLCK 0.1 mM, PMSF 0.05 mM,leupeptin 5 μg/ml). The concentration of cells was approximately3.55×10⁷/ml. The cells were then spun down, resuspended in 1.775 ml 70%NIB-30% glycerol, and frozen as 50 μl aliquots in liquid nitrogen.

Step 2: Membrane permeabilization of Nuclei.

The membrane of nuclei prepared as described in step 1 was permeabilizedusing lysolecithin. Frozen cells were warmed quickly, diluted with NIB,and lysed by addition of lysolecithin at a final concentration of 40μg/ml for 5 minutes at 25° C. At this point the nuclei are surrounded bya cytoskeletal matrix and do not expand or divide if contacted withactivating extract.

Step 3: Removal of cytoskeletal proteins surrounding the nucleus andnuclear matrix proteins within the nucleus.

The nuclei from step 2 were treated with trypsin using variable amountsof enzyme and treatment times. Increasing the length of trypsintreatment from 0-15 minutes, at 25° C., increased the extent of DNAsynthesis after standard CSF pretreatment and replication in activatingextract. Incubation times longer than 15 minutes resulted in decreasedreplication, probably due to nuclear damage and clumping. Optimaltrypsin pretreatment used 0.4 μg/ml trypsin for 15 minutes at 25° C.However, as would be appreciated by one skilled in the art, conditionsfor trypsin treatment may vary depending on how the cells are washed toremove protease inhibitors added during cell preparation and the trypsinincubation temperature. These results confirm that, human cell nuclei,like the Xenopus erythrocyte system, should be prepared for activationusing a controlled proteolytic step.

The lysolecithin-trypsin pretreatment was stopped by adding BSA to0.4%+soybean trypsin inhibitor to a final concentration of 30 μg/mlfollowed by gentle centrifugation. The nuclear pellet was suspended andpelleted once more in 0.4% BSA and then in NIB alone.

Step 4: Further pretreatment with CSF Extract.

Washed nuclei were further pretreated in CSF extract to enhanceactivation. Pretreatment as described in steps 2 and 3 was notsufficient to allow swelling and replication of nuclei usingfrozen/thawed activating extracts. This was attributed to the absence ofMPF activity in frozen/thawed activating extract. Indeed, pretreatmentwith CSF extract substantially increased responsiveness oflysolecithin/trypsin pretreated nuclei.

CSF extract further pretreatments were also preformed using varyinglengths of time and temperature. Frozen/thawed CSF extract weresupplemented with 10 mM creatine phosphate, 10 μg/ml creatinephosphokinase, 80 mM β-glycerol-PO₄, and 0.1 mM CaCl₂. H1 kinase levelsin such extracts were high and stable for several hours.

An incubation for 60 minutes at 25° C. followed by 60 minutes at 4⁰ inCSF extract was found to give the highest amount of activation.Incubations at 25° C. for longer than 60 minutes resulted in lower DNAsynthesis, probably because individual nuclei break up into separatechromosomes. Nuclei in these experiments were at 2000 μl, but a broadrange of concentrations should be equally effective.

Additional experiments carried out using Xenopus erythrocytes suggestthat the 60 minute cold incubation after the 60 minute warm stepincreases the rate of subsequent replication. Possibly, the cold stepdisassembles spindles containing microtubules that form around nucleiduring the warm step.

Thus, the CSF is preferably supplemented with 10 mM creatine phosphate,10 μg/ml creatine phosphokinase, 80 mM β-glycerol-PO₄, and 0.1 mM Ca²⁺,and a treatment regime involving incubation for 60 minutes at 25° C.followed by 60 minutes at 4° C. is used in nuclei further pretreatment.

Step 5: Activation of Erythroid Cell Nuclei.

Nuclear swelling, envelope formation, and replication were carried outon human fetal liver erythroid cell nuclei prepared using the optimalconditions described in steps 1-4 above. The nuclei were activated bydiluting the further pretreated nuclei into nine volumes of preparedactivating extract (prepared from eggs activated for 30 minutes andsupplemented with 10 mM creatine phosphate, and 10 μg/ml creatinephosphokinase, plus 16 μ.M biotinylated-dUTP or 0.2 μCi/μl αP³²-dCTP).The resulting nuclei concentration was about 200 μl.

The following measurement were taken: DNA synthesis was monitored usingextract containing P³²dCTP by gel electrophoresis; histone H1 kinase wasmeasured; and samples, labelled with biotinylated-dUTP were taken forcytological analysis of DNA replication and nuclear envelope breakdown.

DNA synthesis began after a lag of 30 minutes and continued until 120minutes. Little or no round-2 DNA synthesis occurred between 150-180minutes, probably because nuclear envelope breakdown had not takenplace. Second mitosis began between 180-210 minutes as seen by the risein H1 kinase activity, and was accompanied by fragmentation of the DNA.

Analysis of the size of the replicated DNA demonstrated that themolecules were initially very large, but at the time that DNA synthesisstopped (120 minutes) a substantial portion of the DNA was converted toa middle molecular weight (MMW) band of approximately 50 kilobases.Subsequent experiments have demonstrated that formation of this bandcoincides with onset of mitosis, even in the absence of a significanthistone H1 kinase peak, as is the case in this experiment. We believethat the MMW DNA band is an artifact generated by SDS-proteinase Kdisruption of the topoisomerase II-DNA complexes involved in chromosomecondensation. Daughter strand deconcatenation is a mandatory part ofchromosome condensation and of necessity requires Type-II topoisomerase(Topo II) breakage of the replicated chromosome at many sites. Thus webelieve that our in vitro conditions allow the normal G2-like period totake place following the S-phase. Chromosome condensation anddeconcatenation takes place during this period.

The process of DNA fragmentation process is distinct from chromosomecondensation and MMW band formation. Fragmentation appears to occur whennuclei enter mitosis without having completed DNA synthesis, i.e.,premature chromosome condensation.

Cytotological analysis demonstrated that little or no nuclear envelopebreakdown occurred in this experiment at 120-150 minutes, also in accordwith the absence of a histone H1 kinase peak. Extensive nuclear swellingbegan about 30 minutes after incubation. Nuclear envelopes formedbetween 30 and 60 minutes and biotin labelling of new DNA began by 60minutes. The intensity of biotin labelling increased during the S-phase,in keeping with P³² labelling. Maximum swelling with chromatindispersion was reached at about 90 minutes, while some condensation ofchromatin took place at about 90-120 minutes although a small amount ofDNA synthesis was still on going. In the period 120-150 minutes therewas marked chromatin condensation suggesting the onset of mitoticprophase, but nuclear envelope breakdown did not take place.

The 90 minute sample was further analyzed to determine the extent ofsample homogeneity. The first 36 nuclei detected with Hoechst stain werephotographed. In replicated nuclei Texas red staining of biotin bleedsthrough into the blue Hoechst channel turning the nuclei purple. About40% of the nuclei failed to swell and failed to replicate. Almost allremaining nuclei swelled and replicated to the full extent. There werevery few partially replicated nuclei. The inability of some of thenuclei to replicate was attributed to nuclear damage since the responseof carefully prepared frog erythrocyte nuclei is much more homogeneous.

Experiments were also carried out to determine whether nuclear swellingwas dependent on DNA synthesis, by activating nucleic in the presence ofaphidicolin (added at T=0). Biotin labelling confirmed that noreplication took place in the aphidicolin treated nuclei. The resultsdemonstrated that even in the absence of replication many nuclei swelledsignificantly.

Step 6: Formation of mitotic chromosomes.

Swelled and replicated nuclei were treated with Cyclin-

90 or CSF extract. Cyclin-

90 was added to activating extract to obtain prophase mitoticchromosomes, while CSF extract was added to obtain metaphase chromosomesand nuclear envelope breakdown.

Addition of {fraction (1/20)}^(th) volume of cyclin-

90 at T=100 (minutes) caused a slight but real improvement in theclarity of prophase chromosomes observed in T=120 nuclei and thereafterseemed to increase the extent of chromosome condensation. Nuclearenvelope breakdown was observed at T=240 and separate, chromosome-likewere released.

Addition of ½ volume of CSF extract at T=100 caused a rapid extensivecondensation of DNA and disappearance of the nuclear envelope. Thisstate of condensation remained stable until T=240.

Example 13 Activation of Human Sperm Nuclei Using a PPT Pretreatment

This example illustrates the use of a permeabilization-protease-thiolreducing agent (PPT) pretreatment to enhance activation of human spermnuclei. Fresh semen from a healthy male donor was obtained, diluted inan equal volume of yolk test buffer (Jasjey, D. G. and Cohen, M. R.Fertility Sterility 35: 205-212, 1981) and frozen in liquid nitrogen inapproximately 1 ml aliquots containing approximately 1×10⁸ sperm ofwhich approximately 68% were motile.

On the day of the experiment, sperm samples were thawed at roomtemperature and washed twice in ice cold NIB buffer by centrifugation.Sperm were then permeabilized by incubation in 100 μg/ml lysolecithinfor 5 minutes at 25° C. and then treated with 100 μg/ml trypsin (e.g., aprotease) for 10 minutes at the same temperature. Thelysolecithin/trypsin treatment was stopped by the addition of soybeantrypsin inhibitor to 30 μg/ml and dialyzed/lyophilized bovine serumalbumin to 0.4% and then washed by centrifugation. The sperm were thenincubated in a solution of 5 mM dithiothreitol (DTT) (e.g., a thiolreducing agent) in 5 mM in NIB for 60 minutes on ice and thenpost-treated with 1 mM N-ethylmaleimide in NIB for 10 minutes at 25° C.A final wash was carried out in NIB and the nuclei were resuspended at40,000/μl. Two additional aliquots were prepared as described aboveexcept that in one case the DTT was omitted from the 60 minuteincubation in NIB, and in the other case the trypsin was omitted duringthe 10 minute incubation following lysolecithin treatment.

Each of the above three samples were then added at a final concentrationof 4000/μl to a frozen/thawed preparation of high speed “prepared CSFextract” that had been supplemented with 10 mM creatine phosphate, 10μg/ml creatine phosphokinase, 80 mM B-glycerophosphate, and 0.1 mMCaCl₂. Nuclei were incubated for 90 minutes at 25° C. and then for 60minutes at 4° C.

Each of the three CSF extract treated samples were then diluted with 9volumes of a frozen/thawed 25 minute activated egg extract supplementedwith 10 mM creatine phosphate, and 10 μg/ml creatine phosphokinase, plus16 mM biotinylated-dUTP and 16 μM MgCl₂, or plus 0.2 μCi/μl αP³²-dCTP.Each sample was incubated at 25° C. and sampled periodically by eitherfixed staining for biotin incorporation into DNA and photographed, orfractionated on agarose gel and counted for incorporation of labelnucleotides into DNA.

Cytology demonstrated that each of the three treatment regimes resultedin swelling of the sperm nuclear DNA, but only the combinedlysolecithin-trypsin-DTT pretreatment procedure resulted in new nuclearenvelope formation, extensive spherical swelling of the nucleus, and newDNA synthesis. DNA synthesis as determined by P³²-dCTP incorporationalso demonstrated that only the combined lysolecithin-trypsin-DTTpretreatment procedure resulted in significant replication. The combinedlysolecithin-trypsin-DTT pretreatment procedure brought more than a 5fold increase in DNA synthesis than pretreatments with lysolecithin andtrypsin or lysolecithin and DTT.

Additional cytological analysis and in situ hybridization was carriedout using two nucleic acid probes; one to a reiterated sequence on theX-chromosome and one to a single copy sequence on chromosome 18. Thelysolecithin-trypsin-DTT pretreated and activated sperm nuclei wererecovered after 120 minutes of incubation in activating extract in thepresence of biotinylated-dUTP. Two nuclei were stained for total DNA(Hoechst=blue), the X-chromosome (using a digoxygenin-labelledprobe-green), newly synthesized biotinylated DNA (Texas-redstreptavidin=red), and chromosome 18 (using a biotinylatedprobe+Texas-red streptavidin=red dots). The intensity of the Hoechststaining and the biotinylated-dUTP staining in the first nucleus wasless than that in the second nucleus. This demonstrates that firstnucleus had only begun in vitro replication while the second nucleus hadreplicated more completely. In addition, the first nucleus containedonly one copy of the X-chromosome, indicating that the probed region ofthe chromosome had not yet replicated, while the second nucleuscontained two copies of the X-chromosome, indicating that it hadreplicated by this time. In addition, two copies of chromosome 18 weredetected in the first nucleus, suggesting that this probe detects anearly replicating sequence. Detection of chromosome 18 was obscured inthe in the second nucleus by the higher level of incorporatedbiotinylated-dUTP.

These results are consistent with the notion that both thiol reductionand protease induced changes in the sperm cytoskeleton and protaminesare required for displacement of protamines and their replacement bychromatin forming histones in the egg extracts. Chromatin assembly isimportant in the formation of the surrounding nuclear envelope andcompletion of the envelope is important for initiation of DNA synthesis.These results also demonstrated that human sperm cell nuclei which havebeen activated and replicated in vitro can be used for genetic analysis.

Additional experiments demonstrated that the optimized protocoldescribed above also resulted in formation of mitotic chromosomes fromhuman sperm nuclei.

Other embodiments are within the following claims.

1. A method of cloning a non-human mammal, said method comprising thesteps of: (a) incubating a permeabilized cell containing a nucleus in areprogramming extract under conditions that allow the elimination of afactor from said nucleus or the addition of a factor from saidreprogramming extract to said nucleus thereby forming a reprogrammednucleus; (b) transplanting said reprogrammed nucleus into a nucleated oran enucleated egg; and (c) allowing said egg to develop into saidnon-human mammal under direction of genetic information contained in thetransplanted activated nucleus.
 2. The method of claim 1, wherein saidnon-human mammal is complete or substantially complete.
 3. The method ofclaim 1, wherein said mammalian cell is a keratinocyte, erythrocyte,fetal cell, placental cell, red blood cell, white blood cell, orleukocyte.
 4. A method of cloning a non-human mammal, said methodcomprising the steps of: (a) contacting a nucleus with a mitoticmetaphase extract under conditions to allow reprogramming of saidnucleus; (b) transplanting said nucleus into an egg; and (c) allowingsaid egg to develop into said non-human mammal under direction ofgenetic information contained in the transplanted activated nucleus. 5.A composition comprising an enhanced mitotic metaphase extract for thereprogramming of somatic cell nuclei, said extract comprising thecytoplasm from a cell prior to S-phase.
 6. The composition of claim 5,wherein said extract has been supplemented with calcium chloride between0.1 to 0.4 mM.
 7. The composition of claim 5, wherein said extract hasbeen supplemented with beta-glycerol phosphate.
 8. The composition ofclaim 5, wherein said extract has been supplemented with an ATPgenerating system comprising ATP, creatine kinase, and creatinephosphate.